Dual RNA-Sequencing Analysis of Resistant (Pinus pinea) and Susceptible (Pinus radiata) Hosts during Fusarium circinatum Challenge

In e na ional Jou nal o Molecula Sciences A icle Dual RNA-Sequencing Analysis o Resis an (Pinus pinea) and Suscep ible (Pinus adia a) Hos s du ing Fusa ium ci cina um Challenge C is ina Zamo a-Balles e os 1,2,* , Glo ia Pin o 3, Joana Ama al 3, Luis Valledo 4, A u Al es 3, Julio J. Diez 1,2 and Jo ge Ma ín-Ga cía1,2   Ci a ion: Zamo a-Balles e os, C.; Pin o, G.; Ama al, J.; Valledo , L.; Al es, A.; Diez, J.J.; Ma ín-Ga cía, J. Dual RNA-Sequencing Analysis o Resis an (Pinus pinea) and Suscep ible (Pinus adia a) Hos s du ing Fusa ium ci cina um Challenge. In . J. Mol. Sci. 2021,22, 5231. h ps://doi.o g/10.3390/ijms22105231 Academic Edi o : JoséManuel Ga cía Ga ido Recei ed: 23 Ap il 2021 Accep ed: 13 May 2021 Published: 15 May 2021 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2021 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). 1Sus ainable Fo es Managemen Resea ch Ins i u e, Uni e si y o Valladolid—INIA, 34004 Palencia, Spain; julioja ie [email p o ec ed] (J.J.D.); [email p o ec ed] (J.M.-G.) 2Depa men o Vege al P oduc ion and Fo es Resou ces, Uni e si y o Valladolid, 34004 Palencia, Spain 3Cen e o En i onmen al and Ma ine S udies, CESAM, Depa men o Biology, Uni e si y o A ei o, 3810-193 A ei o, Po ugal; [email p o ec ed] (G.P.); [email p o ec ed] (J.A.); a u [email p o ec ed] (A.A.) 4Depa men o O ganisms and Sys ems Biology, Uni e si y o O iedo, 33071 O iedo, Spain; [email p o ec ed] *Co espondence: c is inazamo aballes e [email p o ec ed] Abs ac : Fusa ium ci cina um causes one o he mos impo an diseases o coni e s wo ldwide, he pine pi ch canke (PPC). Howe e , no e ec i e ield in e en ion measu es aiming o con ol o e adica e PPC a e a ailable. Due o he a ia ion in hos gene ic esis ance, he de elopmen o esis an a ie ies is pos ula ed as a iable and p omising s a egy. By using an in eg a ed app oach, his s udy aimed o iden i y di e ences in he molecula esponses and physiological ai s o he highly suscep ible Pinus adia a and he highly esis an Pinus pinea o F. ci cina um a an ea ly s age o in ec ion. Dual RNA-Seq analysis also allowed o e alua e pa hogen beha io when in ec ing each pine species. No signi ican changes in he physiological analysis we e ound upon pa hogen in ec ion, al hough ansc ip ional ep og amming was obse ed mainly in he esis an species. The ansc ip ome p o iling o P. pinea e ealed an ea ly pe cep ion o he pa hogen in ec ion oge he wi h a s ong and coo dina ed de ense ac i a ion h ough he ein o cemen and ligni ica ion o he cell wall, he an ioxidan ac i i y, he induc ion o PR genes, and he biosyn hesis o de ense ho mones. On he con a y, P. adia a had a weake esponse, possibly due o impai ed pe cep ion o he ungal in ec ion ha led o a educed downs eam de ense signaling. Fusa ium ci cina um showed a di e en ansc ip omic p o ile depending on he pine species being in ec ed. While in P. pinea, he pa hogen ocused on he deg ada ion o plan cell walls, ac i e up ake o he plan nu ien s was showed in P. adia a. These indings p esen use ul knowledge o he de elopmen o b eeding p og ams o manage PPC. Keywo ds: dual RNA-Seq; Fusa ium ci cina um;Pinus pinea;Pinus adia a; hos -pa hogen in e ac ion; coni e de ense; disease di e en ial suscep ibili y 1. In oduc ion Pinus adia a is he wo ld’s mos widely plan ed ee o i s economic alue [ 1 ]. In he las wo cen u ies, i s as g ow h and wood quali y ha e sa is ied he inc easing demand o imbe and wood p oduc s wo ldwide. In he No h o Spain, he e a e la ge a eas o P. adia a plan a ions (260,000 ha) whe e his species ep esen s he i s coni e species in olume ha es ed (5 million o m 3 ) [ 2 ]. Un o una ely, he Spanish A lan ic egion has wi nessed a p oli e a ion o ungal diseases ha ha e se e ely a ec ed he plan a ions o P. adia a in he las decades. This coni e is especially suscep ible o diseases such as Do his oma needle bligh (DNB), b own spo needle bligh (BSNB), o pine pi ch canke (PPC), which ha e led o an impo an economic impac [3,4]. In . J. Mol. Sci. 2021,22, 5231. h ps://doi.o g/10.3390/ijms22105231 h ps://www.mdpi.com/jou nal/ijms In . J. Mol. Sci. 2021,22, 5231 2 o 27 The in asi e ungus Fusa ium ci cina um is he causal agen o he PPC, being con- side ed one o he mos impo an pa hogens o coni e s globally [ 5 ]. In ec ed seeds a e he main pa hway o in oduc ion in nu se ies, whe e he pa hogen causes p e- and pos - eme gence damping-o and mo ali y o seedlings [ 6 , 7 ]. In he ield, in ec ed ees su e om s em canke s, dieback, and e en b anches and unks gi dling ha cause ee dea h [ 8 ]. The global dis ibu ion o F. ci cina um is mainly due o globaliza ion in e ms o in e na- ional ade o plan ma e ials and he ailed cu en egula ion [ 9 – 11 ]. Recen ly, i has been disco e ed ha F. ci cina um has he abili y o endophy ically colonizing pine bu also non- coni e ous species [ 12 , 13 ]. This ac , besides making he con ol o he disease especially challenging, highligh s he impo ance o using scien i ic ad ances in he de elopmen o e ec i e s a egies [11]. Once es ablished, no iable in e en ion measu e aiming a he con ol o e adica ion o PPC is a ailable. In his con ex , e o es a ion o he damaged a eas wi h gene ically esis an ma e ial seems o be one o he mos p omising s a egies o educe he impac o his disease [ 14 ]. Al hough P. adia a is he mos suscep ible species o PPC, F. ci cina um can in ec up o 60 di e en species o Pinus,Pseudo suga menziesii, and species in gene a Picea and La ix [ 4 , 15 , 16 ]. Among he hos species, he e is a wide ange o suscep ibili y a ia ion o F. ci cina um [ 6 , 17 ], being he na i e Medi e anean Pinus pinea one o he mos esis an [ 18 , 19 ]. Recen ly, unde an in e go e nmen al amewo k o comba ing PPC (Eu opean COST ac ion FP1406), he suscep ibili y le el o p o enances o se e al Eu opean coni e s poin ed ou ha some in e speci ic gene ic esis ance could be ound [ 7 , 16 , 20 ]. In aspeci ic esis ance, which has been ela ed o en i onmen al g adien s o adap i e p ocesses, has been used o success ul b eeding p og ams [ 21 ]. In addi ion, hyb idiza ion wi h esis an species o popula ions showed sui able esul s [ 22 , 23 ]. In Spain, due o he ban on plan ing suscep ible species in in ec ed a eas (Spanish Royal Dec ee 637/2006 and 65/2010), he use o al e na i e species such as eucalyp us is al eady a ac . In his con ex , u he omics s udies a e equi ed o unde s and he mechanisms unde lying disease o e coming and o achie e esis an geno ypes based on his knowledge. Coni e ees ha e se e al de ense mechanisms ha p o ec hem agains pa hogens. The de ensi e sys em o pines includes mechanical ba ie s such as he induced ac i a ion o auma ic esin duc s and cell wall ein o cemen and chemical de enses, including he p oduc ion o oleo esin e penoids [ 24 ]. The alloca ion o esou ces mus be egula ed by he ee in o de o main ain a balance be ween g ow h and de ense, which is key o ee esis ance [ 25 ]. In u n, ungal pa hogens ha e de eloped sophis ica ed pene a ion, in ec- ion, and coloniza ion s a egies o o e come and supp ess hese de ense mechanisms [ 26 ]. In he las decade, an inc eased numbe o s udies ha e examined he pa hosys ems o se e al o es ees a he ansc ip ional le el [ 27 – 32 ]. The de elopmen o NGS and associa ed bioin o ma ics pipelines ha e made easible he applica ion o high- h oughpu ansc ip omic analysis o unsequenced species, allowing a deep desc ip ion o he an- sc ip ional mechanisms implied in plan ungal de ense [ 33 – 37 ]. Speci ically, some s udies ha e examined he esponse o di e en pine species o F. ci cina um. These included a compa a i e ansc ip omic analysis be ween wo con as ing geno ypes o P. adia a inocula ed by F. ci cina um, whe e di e en ial exp ession o se e al pa hogenesis- ela ed (PR) genes, phospho ylase amily (PFP), and non- ace speci ic disease esis ance (NDR1) genes we e ound associa ed wi h he esis an geno ype [ 38 ]. Mo eo e , some impo an de ense- ela ed genes such as phenylalanine ammonia-lyase (PAL) we e down- egula ed in he highly suscep ible Pinus pa ula a a e y ea ly s age o in ec ion (a e 24 h) [ 39 ]. Visse e al. [ 40 ] ocused hei s udy only on PR genes in bo h P. pa ula and he esis an hos P. ecunumanii a e F. ci cina um in ec ion. Howe e , none o hese s udies examined he ansc ip ome o he pa hogenic ungus upon in ec ion. Cu en sequencing pla o ms gene a e an unp eceden ed numbe o eads, making possible he simul aneous analysis o ansc ip s bo h o pa hogens and hei hos s [ 41 ]. This app oach, called dual RNA-Seq, has been employed o e eal he in e ac ion o Pinus pinas e wi h F. ci cina um [ 42 ]. The mode a e esis ance o P. pinas e was e lec ed in he In . J. Mol. Sci. 2021,22, 5231 3 o 27 ea ly induc ion o genes associa ed wi h a complex phy oho mone signaling o jasmonic acid (JA), e hylene (ET), and salicylic acid (SA). On he o he hand, i was sugges ed a pe u ba ion in he hos phy oho mone homeos asis by F. ci cina um h ough he exp ession o ungal genes ela ed o hese phy oho mones [ 42 ]. Plan ho mones a e impo an egula o s o me abolism being closely ela ed o he plan de ense [ 43 ], esul ing in a complex signaling c oss- alk o phy oho mones such as JA, ET, SA, auxins, cy okinins (CKs), gibbe ellins (GAs), abscisic acid (ABA), and b assinos e oids (BRs). JA and ET a e in ol ed in he ac i a ion o de ense esponses agains nec o ophic pa hogens, while bio ophic and hemibio ophic (as F. ci cina um) pa hogens a e gene ally associa ed wi h SA induc ion and he es ablishmen o sys emic acqui ed esis ance (SAR) [ 44 ]. SA and JA/ET de ense pa hways a e in gene al mu ually an agonis ic, al hough syne gis ic in e ac ions ha e been epo ed as well [ 44 ]. Recen ly, Visse e al. [ 45 ] ocused hei ansc ip omic s udy on he ho mone egula ion in P. pa ula and P. ecunumanii challenged wi h F. ci cina um. In his s udy, a s onge and ea lie esponse was obse ed in he esis an P. ecunumanii h ough he coo dina ion o auxin, ET, JA, and SA signaling,whe eas P. pa ula showed a delayed esponse, which was associa ed wi h i s suscep ibili y o F. ci cina um. In addi ion, he exp ession o genes ela ed o e gos e ol biosyn hesis in F. ci cina um was lowe du ing he in ec ion o he esis an hos , which was poin ed o a highe ulne abili y o he pa hogen o coni e PR1 genes. Physiological and ho monal changes ha e also been in es iga ed in he Pinus-F. ci cina um in e ac ion. While symp oma ic P. adia a and P. pinas e su e ed a wa e dep i a ion-like scena io and pho osyn he ic limi a ion a e F. ci cina um in ec ion, P. pinea opened hei s oma a, inc emen ing he anspi a ion and accumula ed glyce ol [ 19 ]. Fu he mo e, Ama al e al. [ 46 ] sugges ed ha ABA ca abolism could play a key ole in PPC o e come. Gene ac i a ion and ansc ip ion, p o ein and enzyme ac i i ies, p ima y and sec- onda y me abolism, and ho mones and me aboli es signaling a e he main mechanisms ep og ammed in plan s unde pa hogen challenges. To unde s and he p ocesses ha unde lie he esis ance o he Medi e anean species P. pinea and he suscep ibili y o he ypically alloch honous species P. adia a agains he pa hogenic ungus F. ci cina um, a comp ehensi e s udy o he egula ion o he a ay o genes in ol ed in pine de ense mech- anisms has been pe o med. Physiological analyses we e ca ied ou in o de o u he suppo he esul s ob ained a he ansc ip ional le el. This combined ansc ip omic and physiological app oach is expec ed o imp o e he unde s anding o hos -pa hogen in e ac ion and iden i y key playe s and pa hways ha may be use ul o he de elopmen o esis an geno ypes h ough b eeding and/o gene ic enginee ing. 2. Resul s 2.1. Di e ences in he Suscep ibili y Be ween he Hos s A he ime o sampling, none o he seedlings showed symp oms o he disease. In o de o alida e he suscep ibili y o he hos s, 12 seedlings we e moni o ed o each species du ing he 84 days. Inocula ed seedlings o P. adia a began o die 61 days a e inocula ion and, by he end o he expe imen , all o hem had died (Figu e 1). On he con a y, no mo ali y was eco ded o inocula ed P. pinea seedlings. In addi ion, inocula ed P. pinea seedlings did no show symp oms o he disease by he end o he expe imen , indica ing an o e coming o he disease. S a is ically, he su i al analysis e ealed signi ican di e ences be ween bo h species (χ2= 22.7, p< 0.001). In . J. Mol. Sci. 2021,22, 5231 4 o 27 In . J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 o 27 Figu e 1. Su i al p obabili y plo de e mined using he Kaplan–Meie es ima e o he su i al unc ion o P. pinea and P. adia a seedlings inocula ed wi h F. ci cina um. Di e en le e s indica e signi ican p- alues. 2.2. Physiological Measu emen s The wa e po en ial o bo h species was no signi ican ly a ec ed by ungal in ec ion, al hough a sligh educ ion in P. adia a alues and an inc ease in P. pinea can be obse ed (Figu e S1). The analysis con i med ha wa e po en ial is signi ican ly in luenced by he species. Simila ly, he gas exchange and s oma al conduc ance pa ame e s we e only sig- ni ican ly di e en be ween species bu no in luenced by he pa hogen e ec (Figu e 2). The ne CO2 assimila ion a e (A), he s oma al conduc ance (gs), and he anspi a ion a e (E) we e highe in P. pinea, while he needle CO2 concen a ion (Ci) was sligh ly highe in P. adia a. Figu e 2. Needle gas exchange- ela ed pa ame e s o P. pinea and P. adia a inocula ed wi h F. ci ci- na um and con ols a 4 dpi. (A) S oma al conduc ance. (B) T anspi a ion a e. (C) Ne CO2 assimi- la ion a e. (D) Sub-s oma al CO2 concen a ion. E o ba s show he s anda d de ia ion. Di e en le e s abo e he ba s indica e signi ican di e ences (ANOVA, p < 0.05). Figu e 1. Su i al p obabili y plo de e mined using he Kaplan–Meie es ima e o he su i al unc ion o P. pinea and P. adia a seedlings inocula ed wi h F. ci cina um. Di e en le e s indica e signi ican p- alues. 2.2. Physiological Measu emen s The wa e po en ial o bo h species was no signi ican ly a ec ed by ungal in ec ion, al hough a sligh educ ion in P. adia a alues and an inc ease in P. pinea can be obse ed (Figu e S1). The analysis con i med ha wa e po en ial is signi ican ly in luenced by he species. Simila ly, he gas exchange and s oma al conduc ance pa ame e s we e only signi ican ly di e en be ween species bu no in luenced by he pa hogen e ec ( Figu e 2 ). The ne CO 2 assimila ion a e (A), he s oma al conduc ance (gs), and he anspi a ion a e (E) we e highe in P. pinea, while he needle CO 2 concen a ion (Ci) was sligh ly highe in P. adia a. In . J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 o 27 Figu e 1. Su i al p obabili y plo de e mined using he Kaplan–Meie es ima e o he su i al unc ion o P. pinea and P. adia a seedlings inocula ed wi h F. ci cina um. Di e en le e s indica e signi ican p- alues. 2.2. Physiological Measu emen s The wa e po en ial o bo h species was no signi ican ly a ec ed by ungal in ec ion, al hough a sligh educ ion in P. adia a alues and an inc ease in P. pinea can be obse ed (Figu e S1). The analysis con i med ha wa e po en ial is signi ican ly in luenced by he species. Simila ly, he gas exchange and s oma al conduc ance pa ame e s we e only sig- ni ican ly di e en be ween species bu no in luenced by he pa hogen e ec (Figu e 2). The ne CO2 assimila ion a e (A), he s oma al conduc ance (gs), and he anspi a ion a e (E) we e highe in P. pinea, while he needle CO2 concen a ion (Ci) was sligh ly highe in P. adia a. Figu e 2. Needle gas exchange- ela ed pa ame e s o P. pinea and P. adia a inocula ed wi h F. ci ci- na um and con ols a 4 dpi. (A) S oma al conduc ance. (B) T anspi a ion a e. (C) Ne CO2 assimi- la ion a e. (D) Sub-s oma al CO2 concen a ion. E o ba s show he s anda d de ia ion. Di e en le e s abo e he ba s indica e signi ican di e ences (ANOVA, p < 0.05). Figu e 2. Needle gas exchange- ela ed pa ame e s o P. pinea and P. adia a inocula ed wi h F. ci cina um and con ols a 4 dpi. ( A ) S oma al conduc ance. ( B ) T anspi a ion a e. ( C ) Ne CO 2 assimila ion a e. ( D ) Sub-s oma al CO 2 concen a ion. E o ba s show he s anda d de ia ion. Di e en le e s abo e he ba s indica e signi ican di e ences (ANOVA, p< 0.05). In . J. Mol. Sci. 2021,22, 5231 5 o 27 2.3. Dual RNA-Sequencing S a is ics The cha ac e iza ion and compa ison o P. pinea and P. adia a esponses o F. ci cina um in ec ion and he simul aneous cha ac e iza ion o he F. ci cina um gene exp ession we e ca ied ou using a dual RNA-Seq app oach. An a e age o 51.8 ± 4.41% o P. pinea eads, and 82.9 ± 1.84% o P. adia a eads we e mapped o he e e ence genome o P. aeda (Table S1). Conside ing he in ec ed samples, o P. pinea and P. adia a, espec i ely, an a e age o 1.86 ± 2.33% and 0.57 ± 0.29% eads mapped o he F. ci cina um genome. The PCA allowed iden i ying wo sample ou lie s in he con ol ea men s o each pine species ha we e disca ded om he downs eam analysis (Figu e S2). Once he a ypical samples we e emo ed, he i s p incipal componen in he hos analysis showed a s ong sepa a ion by pine species. On he second p incipal componen , he di e en ea men s (in ec ed by F. ci cina um and con ol) we e clus e ed in sepa a ed g oups, being mo e e iden be ween he P. pinea ea men s (Figu e 3A). The isualiza ion o he F. ci cina um exp ession analysis wi h PCA showed a clea sepa a ion acco ding o pine species in ec ed (Figu e 3B). In . J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 o 27 2.3. Dual RNA-Sequencing S a is ics The cha ac e iza ion and compa ison o P. pinea and P. adia a esponses o F. ci cina- um in ec ion and he simul aneous cha ac e iza ion o he F. ci cina um gene exp ession we e ca ied ou using a dual RNA-Seq app oach. An a e age o 51.8 ± 4.41% o P. pinea eads, and 82.9 ± 1.84% o P. adia a eads we e mapped o he e e ence genome o P. aeda (Table S1). Conside ing he in ec ed samples, o P. pinea and P. adia a, espec i ely, an a e age o 1.86 ± 2.33% and 0.57 ± 0.29% eads mapped o he F. ci cina um genome. The PCA allowed iden i ying wo sample ou lie s in he con ol ea men s o each pine spe- cies ha we e disca ded om he downs eam analysis (Figu e S2). Once he a ypical sam- ples we e emo ed, he i s p incipal componen in he hos analysis showed a s ong sepa a ion by pine species. On he second p incipal componen , he di e en ea men s (in ec ed by F. ci cina um and con ol) we e clus e ed in sepa a ed g oups, being mo e e iden be ween he P. pinea ea men s (Figu e 3A). The isualiza ion o he F. ci cina um exp ession analysis wi h PCA showed a clea sepa a ion acco ding o pine species in ec ed (Figu e 3B). Figu e 3. Two-dimensional sca e plo o he p incipal componen analyses (PCA) o (A) Pinus adia a and P. pinea di e en ea men s and (B) Fusa ium ci cina um in ec ing e e y hos species. The dis ances app oxima e he ypical log2 old changes be ween he samples. Fo he hos da ase , ansc ip s wi h coun s pe million (CPM) > 1 in a leas h ee lib a ies ( he minimum numbe o lib a ies among ea men s) we e conside ed easona- ble o use in u he compa isons. In he case o he pa hogen da ase , only genes p esen in a leas ou eplica es wi h CPM > 2 we e used o DE analysis. A o al o 20,380 genes we e abo e abundance and consis ency be ween eplica es h esholds in he hos s and 8866 genes in he pa hogen. DE analysis o P. pinea (inocula ed s. con ol) iden i ied 1822 DEGs, whe eas 371 signi ican DEGs we e iden i ied o P. adia a (Table 1; Tables S2 and S3; Figu e S3). Only 249 up- egula ed DEGs ou o 1417 we e sha ed in he de ense e- sponse be ween bo h species, wi hou sha ing any o he ep essed genes (Figu e 4). Fusa ium ci cina um DE analysis o inocula ed samples (P. pinea s. P. adia a) iden i ied 204 signi ican DEGs (Table 1; Table S4; Figu e S4). Figu e 3. Two-dimensional sca e plo o he p incipal componen analyses (PCA) o ( A )Pinus adia a and P. pinea di e en ea men s and ( B )Fusa ium ci cina um in ec ing e e y hos species. The dis ances app oxima e he ypical log2 old changes be ween he samples. Fo he hos da ase , ansc ip s wi h coun s pe million (CPM) > 1 in a leas h ee lib a ies ( he minimum numbe o lib a ies among ea men s) we e conside ed easonable o use in u he compa isons. In he case o he pa hogen da ase , only genes p esen in a leas ou eplica es wi h CPM > 2 we e used o DE analysis. A o al o 20,380 genes we e abo e abundance and consis ency be ween eplica es h esholds in he hos s and 8866 genes in he pa hogen. DE analysis o P. pinea (inocula ed s. con ol) iden i ied 1822 DEGs, whe eas 371 signi ican DEGs we e iden i ied o P. adia a (Table 1; Tables S2 and S3; Figu e S3). Only 249 up- egula ed DEGs ou o 1417 we e sha ed in he de ense esponse be ween bo h species, wi hou sha ing any o he ep essed genes (Figu e 4). Fusa ium ci cina um DE analysis o inocula ed samples (P. pinea s. P. adia a) iden i ied 204 signi ican DEGs (Table 1; Table S4; Figu e S4). The esul s o he combined anno a ion o F. ci cina um h ough he EnTAP pipeline yielded 9757 ansc ip s wi h coding egions p edic ed using GeneMa kS-T (Table S5). The bes -hi selec ion o BLASTp, which ep esen s he mos in o ma i e ma ch a ailable, combining he hi s om each da abase wi hin he co e age and e- alue h esholds speci ied, esul ed in alignmen s o 9406 (78%) p o eins. The anno a ed ansc ip s we e mos ly sha ed wi h Fusa ium species. EggNOG anno a ion assigned 9527 (97%) F. ci cina um sequences o amilies, and In e P oScan anno a ion assigned 6803 (70%) o domains. To sum up, a o al o 9650 (99%) sequences we e anno a ed, o which 7434 (76%) and 2494 (26%) we e assigned gene on ology (GO) e ms and KEGG pa hways, espec i ely. To iden i y pu a i e genes in ol ed in pa hogenici y, we analyzed he F. ci cina um sequences o simila i y wi h subjec s in he pa hogen-hos in e ac ion gene da abase (PHI-base), esul ing in 2489 (26%) alignmen s (Table S6). In . J. Mol. Sci. 2021,22, 5231 6 o 27 Table 1. Summa y o di e en ially exp essed genes iden i ied in P. pinea and P. adia a o each compa ison. O ganism Genes Up-Regula ed 1Genes Down-Regula ed 1 Di e en ially exp essed hos genes 2 P. pinea 1307 515 P. adia a 359 12 Di e en ially exp essed pa hogen genes 3 F. ci cina um 118 86 1Numbe o up- egula ed and down- egula ed signi ican ly di e en ially exp essed genes ( alse disco e y a e, FDR < 0.05 and a log 2 ( old change) > 0.5 o < − 0.5) using he QLTes (Benjamini and Hochbe g FDR co ec ion) wi h edgeR. 2 Hos genes di e en ially exp essed in inocula ed ela i e o mock-inocula ed hos exp ession da a. 3 Di e en ially exp essed F. ci cina um genes in P. pinea ela i e o P. adia a inocula ed samples om pa hogen exp ession da a. In . J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 o 27 Table 1. Summa y o di e en ially exp essed genes iden i ied in P. pinea and P. adia a o each compa ison. O ganism Genes Up-Regula ed 1 Genes Down-Regula ed 1 Di e en ially exp essed hos genes 2 P. pinea 1307 515 P. adia a 359 12 Di e en ially exp essed pa hogen genes 3 F. ci cina um 118 86 1 Numbe o up- egula ed and down- egula ed signi ican ly di e en ially exp essed genes ( alse disco e y a e, FDR < 0.05 and a log2( old change) > 0.5 o < −0.5) using he QLTes (Benjamini and Hochbe g FDR co ec ion) wi h edgeR. 2 Hos genes di e en ially exp essed in inocula ed ela i e o mock-inocula ed hos exp ession da a. 3 Di e en ially exp essed F. ci cina um genes in P. pinea ela i e o P. adia a inocula ed samples om pa hogen exp ession da a. Figu e 4. Venn diag am showing he numbe o P. pinea and P. adia a genes up- egula ed (A) and down- egula ed (B). The esul s o he combined anno a ion o F. ci cina um h ough he EnTAP pipeline yielded 9757 ansc ip s wi h coding egions p edic ed using GeneMa kS-T (Table S5). The bes -hi selec ion o BLASTp, which ep esen s he mos in o ma i e ma ch a ailable, combining he hi s om each da abase wi hin he co e age and e- alue h esholds speci- ied, esul ed in alignmen s o 9406 (78%) p o eins. The anno a ed ansc ip s we e mos ly sha ed wi h Fusa ium species. EggNOG anno a ion assigned 9527 (97%) F. ci cina- um sequences o amilies, and In e P oScan anno a ion assigned 6803 (70%) o domains. To sum up, a o al o 9650 (99%) sequences we e anno a ed, o which 7434 (76%) and 2494 (26%) we e assigned gene on ology (GO) e ms and KEGG pa hways, espec i ely. To iden i y pu a i e genes in ol ed in pa hogenici y, we analyzed he F. ci cina um se- quences o simila i y wi h subjec s in he pa hogen-hos in e ac ion gene da abase (PHI- base), esul ing in 2489 (26%) alignmen s (Table S6). 2.4. O e -Rep esen ed Gene On ologies and KEGG Pa hways in Hos Da ase s Challenged Wi h Fusa ium ci cina um The DEGs in he hos da ase s we e analyzed o iden i y o e - ep esen a ion o GO e ms ocusing on biological p ocesses (BP) and molecula unc ion (MF), as well as o KEGG pa hways (p- alue < 0.05; Tables S7–S10). This analysis has shown he wide ange o biological p ocesses unde lying each hos species in esponse o F. ci cina um. The up and down- egula ed gene da ase s o P. pinea sha ed he majo i y o en iched GO e ms and we e ela ed o plan de ense p ocesses such as oxida ion- educ ion, la onoid bio- syn hesis and me abolism, e hylene, and jasmonic acid biosyn hesis, egula ion o an ho- cyanin me abolism, and oxylipin biosyn hesis. No GO e ms o KEGG pa hways we e en iched in he down- egula ed da ase o P. adia a DEGs, bu a la ge numbe o GO Figu e 4. Venn diag am showing he numbe o P. pinea and P. adia a genes up- egula ed ( A ) and down- egula ed (B). 2.4. O e -Rep esen ed Gene On ologies and KEGG Pa hways in Hos Da ase s Challenged Wi h Fusa ium ci cina um The DEGs in he hos da ase s we e analyzed o iden i y o e - ep esen a ion o GO e ms ocusing on biological p ocesses (BP) and molecula unc ion (MF), as well as o KEGG pa hways (p- alue < 0.05; Tables S7–S10). This analysis has shown he wide ange o biological p ocesses unde lying each hos species in esponse o F. ci cina um. The up and down- egula ed gene da ase s o P. pinea sha ed he majo i y o en iched GO e ms and we e ela ed o plan de ense p ocesses such as oxida ion- educ ion, la onoid biosyn hesis and me abolism, e hylene, and jasmonic acid biosyn hesis, egula ion o an hocyanin me abolism, and oxylipin biosyn hesis. No GO e ms o KEGG pa hways we e en iched in he down- egula ed da ase o P. adia a DEGs, bu a la ge numbe o GO e ms we e en iched in he up- egula ed DEGs. Mos o hem we e also ound o be en iched in he P. pinea da ase s (Figu e 5). The majo i y o en iched KEGG pa hways we e ound in bo h up- and down- egula ed da ase s o P. pinea. The pa hways uniquely en iched in he up- egula ed genes we e ela ed o plan de ense, such as plan ho mone signal ansduc ion, zea in biosyn hesis, L-asco ba e oxidase ac i i y, and ubiquinone and o he e penoid-quinone biosyn hesis; con as ing wi h he exclusi e down- egula ion o genes ela ed o he ene gy me abolism o he plan . The mos en iched KEGG pa hways o he up- egula ed DEGs in P. adia a we e seconda y me aboli es biosyn hesis and plan s ci cadian hy hm pa hway. O e - ep esen ed da ase s o bo h hos s sha ed mos o he KEGG pa hways as well, al hough he e we e mo e pa hways unique o he esis an hos han o he suscep ible species. In . J. Mol. Sci. 2021,22, 5231 7 o 27 In . J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 o 27 e ms we e en iched in he up- egula ed DEGs. Mos o hem we e also ound o be en- iched in he P. pinea da ase s (Figu e 5). Figu e 5. Rep esen a ion o he mos signi ican ly (p < 0.05) en iched GO e ms (biological p o- cesses) o he up- egula ed genes o P. pinea (blue ba s) and P. adia a (g een ba s) in ec ed by F. ci cina um. The majo i y o en iched KEGG pa hways we e ound in bo h up- and down- egu- la ed da ase s o P. pinea. The pa hways uniquely en iched in he up- egula ed genes we e ela ed o plan de ense, such as plan ho mone signal ansduc ion, zea in biosyn hesis, L-asco ba e oxidase ac i i y, and ubiquinone and o he e penoid-quinone biosyn hesis; con as ing wi h he exclusi e down- egula ion o genes ela ed o he ene gy me abolism o he plan . The mos en iched KEGG pa hways o he up- egula ed DEGs in P. adia a we e seconda y me aboli es biosyn hesis and plan s ci cadian hy hm pa hway. O e - ep esen ed da ase s o bo h hos s sha ed mos o he KEGG pa hways as well, al hough he e we e mo e pa hways unique o he esis an hos han o he suscep ible species. 2.5. Exp ession o Genes In ol ed in Pinus Response o Fusa ium ci cina um In ec ion The in ec ion by F. ci cina um has caused se e e ansc ip ional ep og amming in bo h pine species. Se e al o hese genes belong o unc ional g oups in ol ed in plan de ense agains pa hogens, such as signal pe cep ion and ansc ip ional egula ion, sec- onda y me abolism, an imic obial ac i i y, cell wall ein o cemen and ligni ica ion, and pa hogenesis- ela ed genes. A close look a he DEGs in each hos species e ealed some di e ences in esponse o F. ci cina um ega ding hese p ocesses (Table S11), which could be key o pine esis ance o PPC. Belonging o he pa e n ecogni ion ecep o (PRR) amily, di e en classes o ecep- o kinases such as leucine- ich epea (LRR) ecep o -like kinases (RLKs), se ine/ h eo- nine-p o ein kinases (STKs), and cys eine- ich ecep o -like p o ein kinases (CRKs) we e highly abundan among he DEG in P. pinea; while only se en we e up- egula ed in P. adia a (Figu e 6). Se e al wall-associa ed ecep o kinase-like (WAKL) genes we e mainly Figu e 5. Rep esen a ion o he mos signi ican ly (p< 0.05) en iched GO e ms (biological p ocesses) o he up- egula ed genes o P. pinea (blue ba s) and P. adia a (g een ba s) in ec ed by F. ci cina um. 2.5. Exp ession o Genes In ol ed in Pinus Response o Fusa ium ci cina um In ec ion The in ec ion by F. ci cina um has caused se e e ansc ip ional ep og amming in bo h pine species. Se e al o hese genes belong o unc ional g oups in ol ed in plan de ense agains pa hogens, such as signal pe cep ion and ansc ip ional egula ion, sec- onda y me abolism, an imic obial ac i i y, cell wall ein o cemen and ligni ica ion, and pa hogenesis- ela ed genes. A close look a he DEGs in each hos species e ealed some di e ences in esponse o F. ci cina um ega ding hese p ocesses (Table S11), which could be key o pine esis ance o PPC. Belonging o he pa e n ecogni ion ecep o (PRR) amily, di e en classes o ecep o kinases such as leucine- ich epea (LRR) ecep o -like kinases (RLKs), se ine/ h eonine- p o ein kinases (STKs), and cys eine- ich ecep o -like p o ein kinases (CRKs) we e highly abundan among he DEG in P. pinea; while only se en we e up- egula ed in P. adia a (Figu e 6). Se e al wall-associa ed ecep o kinase-like (WAKL) genes we e mainly up- egula ed in P. pinea, whe eas only one did in P. adia a. Fu he mo e, 12 genes encoding lec in domain-con aining ecep o kinase p o eins, which ha e a ole in chi in-con aining pa hogen ecogni ion, we e exclusi ely up- egula ed in he esis an species (Figu e 6). As a p o ec i e mechanism, wo se ine/ h eonine phospha ases ype 2A (PP2A) we e down- egula ed in P. pinea. PRR-de i ed signals igge mi ogen-ac i a ed p o ein kinases (MAPK) cascades and calcium-dependen p o ein kinases (CDPKs). Se e al mi ogen- ac i a ed p o ein kinase kinase (MAPKK) and mi ogen-ac i a ed p o ein kinase kinase kinase (MAPKKK) we e up- egula ed in P. pinea (Figu e 6). Genes ela ed o calcium (Ca 2+ ) signaling, including calmodulin and calcineu in B–like (CBL) in e ac ing p o ein kinases (CIPKs), we e up and down- egula ed in P. pinea. Pa icula ly, wo genes encoding CIPK3 p o ein kinases, which a e implica ed in s ess and ABA esponses, we e up- egula ed. In . J. Mol. Sci. 2021,22, 5231 8 o 27 In . J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 8 o 27 up- egula ed in P. pinea, whe eas only one did in P. adia a. Fu he mo e, 12 genes encod- ing lec in domain-con aining ecep o kinase p o eins, which ha e a ole in chi in-con ain- ing pa hogen ecogni ion, we e exclusi ely up- egula ed in he esis an species (Figu e 6). As a p o ec i e mechanism, wo se ine/ h eonine phospha ases ype 2A (PP2A) we e down- egula ed in P. pinea. PRR-de i ed signals igge mi ogen-ac i a ed p o ein kinases (MAPK) cascades and calcium-dependen p o ein kinases (CDPKs). Se e al mi ogen-ac- i a ed p o ein kinase kinase (MAPKK) and mi ogen-ac i a ed p o ein kinase kinase ki- nase (MAPKKK) we e up- egula ed in P. pinea (Figu e 6). Genes ela ed o calcium (Ca2+) signaling, including calmodulin and calcineu in B–like (CBL) in e ac ing p o ein kinases (CIPKs), we e up and down- egula ed in P. pinea. Pa icula ly, wo genes encoding CIPK3 p o ein kinases, which a e implica ed in s ess and ABA esponses, we e up- egula ed. Figu e 6. Di e en ially exp essed genes (DEGs) wi h a ole in signal pe cep ion. Numbe s ep e- sen he log2 old change alue based on he compa ison o he ansc ip le els be ween he pine seedlings in ec ed by F. ci cina um and mock-inocula ed con ol. G oups o genes a e abb e ia ed as ollows: RLKs, ecep o -like kinases; LecRLKs, lec in domain-con aining ecep o kinase; STKs, se ine/ h eonine-p o ein kinases; LRR-RL ST, ecep o -like se ine/ h eonine-p o ein kinase wi h leucine- ich epea domain; STPK HT1-like, se ine h eonine-p o ein kinase HT1-like; WAKL, wall-associa ed ecep o kinase-like; LRR-RLKs, leucine- ich epea ecep o -like p o ein kinases; CRKs, cys eine- ich ecep o -like p o ein kinase; MAPKK, mi ogen-ac i a ed p o ein kinase ki- nase; MAPKKK, mi ogen-ac i a ed p o ein kinase kinase kinase; PP2A, se ine/ h eonine phospha- ases ype 2A. A la ge numbe o genes ha encode R p o eins we e mainly up- egula ed in P. pinea; while only one was up- egula ed in he suscep ible species and ano he one was highly down- egula ed (|log2[Fold Change]| = 6.6). WRKY ansc ip ion ac o s (WRKY75, 72, 51, 14, 59, and 17) we e induced in he DEGs o he esis an species. Only one WRKY75 was weakly up- egula ed in P. adia a. An e hylene esponse ac o (ERF) was exp essed by bo h species, his ime, o a g ea e ex en in he P. adia a. Membe s o o he amilies o ansc ip ion ac o s such as MYB and bZIP we e up- and down- egula ed in P. pinea. Figu e 6. Di e en ially exp essed genes (DEGs) wi h a ole in signal pe cep ion. Numbe s ep- esen he log 2 old change alue based on he compa ison o he ansc ip le els be ween he pine seedlings in ec ed by F. ci cina um and mock-inocula ed con ol. G oups o genes a e abb e- ia ed as ollows: RLKs, ecep o -like kinases; LecRLKs, lec in domain-con aining ecep o kinase; STKs, se ine/ h eonine-p o ein kinases; LRR-RL ST, ecep o -like se ine/ h eonine-p o ein kinase wi h leucine- ich epea domain; STPK HT1-like, se ine h eonine-p o ein kinase HT1-like; WAKL, wall-associa ed ecep o kinase-like; LRR-RLKs, leucine- ich epea ecep o -like p o ein kinases; CRKs, cys eine- ich ecep o -like p o ein kinase; MAPKK, mi ogen-ac i a ed p o ein kinase kinase; MAPKKK, mi ogen-ac i a ed p o ein kinase kinase kinase; PP2A, se ine/ h eonine phospha ases ype 2A. A la ge numbe o genes ha encode R p o eins we e mainly up- egula ed in P. pinea; while only one was up- egula ed in he suscep ible species and ano he one was highly down- egula ed (|log 2 [Fold Change]| = 6.6). WRKY ansc ip ion ac o s (WRKY75, 72, 51, 14, 59, and 17) we e induced in he DEGs o he esis an species. Only one WRKY75 was weakly up- egula ed in P. adia a. An e hylene esponse ac o (ERF) was exp essed by bo h species, his ime, o a g ea e ex en in he P. adia a. Membe s o o he amilies o ansc ip ion ac o s such as MYB and bZIP we e up- and down- egula ed in P. pinea. The p oduc ion o seconda y me aboli es h ough he phenylp opanoid pa hway is essen ial o plan de ense [ 47 ] as i supplies p ecu so s o lignin biosyn hesis. Pine species exhibi ed a di e en ial esponse o genes ela ed o he biosyn hesis o phenolic compounds (Table S11; Figu e 7). This pa hway was widely induced upon pa hogen in ec ion in P. pinea, and induc ion o genes encoding leucoan hocyanidin educ ase (LAR) and alkaloid be be ine p o eins was obse ed in bo h species. Rega ding iso la onoids biosyn hesis, se e al iso la one educ ases (IFRs) we e up- egula ed in P. pinea. A conside able numbe o p edic ed e pene syn hases we e up- egula ed in P. pinea, wi h only hal o hem p esen in P. adia a, mos o hem uniquely in hese samples. O he pa hogenesis- ela ed p o eins wi h an i ungal ac i i y, such as cys eine- ich sec e o y p o eins, we e up- egula ed in bo h species (log2[Fold Change] o 8 and 10). In . J. Mol. Sci. 2021,22, 5231 9 o 27 In . J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 9 o 27 The p oduc ion o seconda y me aboli es h ough he phenylp opanoid pa hway is essen ial o plan de ense [47] as i supplies p ecu so s o lignin biosyn hesis. Pine species exhibi ed a di e en ial esponse o genes ela ed o he biosyn hesis o phenolic com- pounds (Table S11; Figu e 7). This pa hway was widely induced upon pa hogen in ec ion in P. pinea, and induc ion o genes encoding leucoan hocyanidin educ ase (LAR) and al- kaloid be be ine p o eins was obse ed in bo h species. Rega ding iso la onoids biosyn- hesis, se e al iso la one educ ases (IFRs) we e up- egula ed in P. pinea. A conside able numbe o p edic ed e pene syn hases we e up- egula ed in P. pinea, wi h only hal o hem p esen in P. adia a, mos o hem uniquely in hese samples. O he pa hogenesis- ela ed p o eins wi h an i ungal ac i i y, such as cys eine- ich sec e o y p o eins, we e up- egula ed in bo h species (log2[Fold Change] o 8 and 10). Figu e 7. Simpli ied scheme o he la onoid and lignin biosyn he ic pa hway. Some c i ical up- egula ed (g een) o down- egula ed ( ed) enzymes o P. pinea a e indica ed and abb e ia ed as ollows: PAL, phenylalanine ammonia-lyase; C4H, cinnama e 4-hyd oxylase; 4CL, 4-couma a e: CoA ligase; CHS, chalcone syn hase; CHI, la anone isome ase; F3H, Fla anone 3-hyd oxylase; DFR, dihyd o la onol 4- educ ase; LDOX, leucoan hocyanidin dioxygenase; OMT, O-me hyl ans- e ase; UFGT, UDP-glucose: la onoid 3-O-glucosyl ans e ase; RT, pu a i e hamnosyl ans e - ase; CCoAOMT, ca eoyl-CoA O-me hyl ans e ase; CCR, cinnamoyl-CoA educ ase; CAD, cin- namyl alcohol dehyd ogenase; FLS, la onol syn hase; LAR, leucan hocyanidin educ ase. Indi id- ual di e en ially exp essed genes (DEGs) wi h anno a ed unc ions a e lis ed along he Y-axis o P. pinea and P. adia a. The ba s in he X-axis ep esen he le el o log2 old change alue based on he compa ison o he ansc ip le els be ween he pine seedlings in ec ed by F. ci cina um and mock-inocula ed con ol. Red ba s deno e he le el o down- egula ion, and g een ba s indica e he le el o up- egula ion. The DE o genes encoding o cell wall modi ying enzymes o bo h pines is shown in Figu e 8. In e es ingly, a con as ing scena io is shown in he pec ines e ase (PE) and pec- in me hyles e ase inhibi o (PMEI) genes: while 21 genes encoding o PMEI we e highly induced (up o 10.9 log2[ old change]) in P. pinea, only one did in P. adia a, and ano he Figu e 7. Simpli ied scheme o he la onoid and lignin biosyn he ic pa hway. Some c i ical up- egula ed (g een) o down- egula ed ( ed) enzymes o P. pinea a e indica ed and abb e ia ed as ollows: PAL, phenylalanine ammonia-lyase; C4H, cinnama e 4-hyd oxylase; 4CL, 4-couma a e: CoA ligase; CHS, chalcone syn hase; CHI, la anone isome ase; F3H, Fla anone 3-hyd oxylase; DFR, dihy- d o la onol 4- educ ase; LDOX, leucoan hocyanidin dioxygenase; OMT,O-me hyl ans e ase; UFGT, UDP-glucose: la onoid 3-O-glucosyl ans e ase; RT, pu a i e hamnosyl ans e ase; CCoAOMT, ca eoyl-CoA O-me hyl ans e ase; CCR, cinnamoyl-CoA educ ase; CAD, cinnamyl alcohol dehy- d ogenase; FLS, la onol syn hase; LAR, leucan hocyanidin educ ase. Indi idual di e en ially exp essed genes (DEGs) wi h anno a ed unc ions a e lis ed along he Y-axis o P. pinea and P. adia a. The ba s in he X-axis ep esen he le el o log 2 old change alue based on he compa ison o he ansc ip le els be ween he pine seedlings in ec ed by F. ci cina um and mock-inocula ed con ol. Red ba s deno e he le el o down- egula ion, and g een ba s indica e he le el o up- egula ion. The DE o genes encoding o cell wall modi ying enzymes o bo h pines is shown in Figu e 8. In e es ingly, a con as ing scena io is shown in he pec ines e ase (PE) and pec in me hyles e ase inhibi o (PMEI) genes: while 21 genes encoding o PMEI we e highly induced (up o 10.9 log 2 [ old change]) in P. pinea, only one did in P. adia a, and ano he was ep essed. Addi ionally, wo PE we e down- egula ed only in P. pinea. Fu he mo e, many genes ela ed o lignin biosyn hesis we e up- egula ed in P. pinea, being his numbe much smalle in P. adia a (Figu e 7). The exp ession p o ile o DEGs encoding anspo e s demons a ed di e ences in in ec ion esponse be ween hos species. Genes encoding pleio opic d ug esis ance (PDR)- ype ABC anspo e s, mul id ug esis ance-associa ed p o eins (MRP)- ype ABC anspo e s, and o he ABC anspo e s we e up- egula ed in P. pinea; in con as , only one (PDR)- ype ABC anspo e was up- egula ed in P. adia a. Simila ly, se e al genes encoding chlo oplas memb ane-localized membe s o he mul id ug and oxin (MATE) anspo e s we e up- egula ed in P. pinea, bu only one in P. adia a. O he anspo e amilies, including aluminum-ac i a ed mala e, amino acid, ammonium, bidi ec ional suga , lysine/his idine, ni a e, and po assium anspo e s, we e also di e en ially eg- In . J. Mol. Sci. 2021,22, 5231 16 o 27 in bo h pine species. The exp ession o his ansc ip ion ac o is induced e y apidly and egula es bo h s ess ole ance and g ow h inhibi ion [ 66 ], playing a posi i e ole in A abidiopsis de ense agains Bo y is cine ea [ 67 ]. Addi ionally, ERFs may be in ol ed in he supp ession o SA-media ed signaling and SA esponsi e gene PR1. Al hough P. adia a p esen ed a 12- old change o PR1 gene exp ession, wo PAD4 we e down- egula ed in his species, which could e lec he an agonism o SA and JA/ET pa hways. On he o he hand, he esul s showed a mode a e ac i a ion o SA signaling in P. pinea oge he wi h he high up- egula ion o wo PR1 genes. Induc ion o PR1 genes upon F. ci cina um in ec ion has also been obse ed in P. pinas e and in he esis an geno ype o P. adia a [ 38 , 42 ]. In addi ion, he syne gis ic coope a ion o SA and JA has been ex ensi ely epo ed [ 42 , 45 , 68 ]. Ama al e al. [ 46 ] showed ha while he suscep ible P. adia a su e ed pho osyn hesis impai men once PPC symp oms occu , P. pinea was able o main ain i s pho osyn he ic ac i i y o e ime a e F. ci cina um inocula ion. A he ansc ip ional le el, he down- egula ion o genes encoding RuBisCo, pho osys em I eac ion cen e subuni o chlo ophyll a/b binding p o ein (Table S19), due possibly o he highe le els o e hylene [ 69 ], could sugges a educ ion in pho osyn hesis ac i i y in P. pinea. Howe e , his was no e lec ed in he physiological analysis esul s. On he o he hand, he pho osyn hesis in P. adia a was al e ed nei he a he ansc ip ional le el no in he physiological analysis. This highligh s he impo ance o an in eg a ed s udy o ansc ip omic and physiological analysis ha conside s he pos - ansc ip ional egula ion. The gene al s oma a opening and inc eased anspi a ion a e e i ied in P. pinea upon inocula ion wi h F. ci cina um in Ama al e al. [ 46 ] a e a odds wi h ou esul s. The absence o hese changes in ou s udy may be explained by he di e en plan g owing condi ions o plan p o enances. A la ge numbe o p edic ed genes ela ed o cell wall ein o cemen and ligni ica- ion we e induced in he esis an species, oge he wi h a s ong egula ion o cell wall modi ica ion h ough inhibi ion o all exp essed cellulose syn hase p o eins, expansins, and some xyloglucan endo- ansglycosylase (XET) p o eins. The supp ession o cellu- lose syn hesis has been p e iously associa ed wi h enhanced esis ance o ungal and bac e ial pa hogens [ 70 ]. Likewise, he down- egula ion o expansins s eng hens he cell wall by a oiding he loosening o he wall by cell ex ension. In e es ingly, a p edic ed hyd oxyp oline- ich glycop o ein (HRGP) was only up- egula ed in P. pinea, being hese s uc u al p o eins induced in disease- esis an esponses [ 71 ]. The ac ha some genes p edic ed o encode XET p o eins we e down- egula ed could be ela ed o he GO e ms en iched in he up- egula ed DEGs o F. ci cina um ha we e associa ed wi h he deg ada- ion o he cell wall componen s such as polysaccha ide, hemicellulose, and xyloglucan. In ac , i has been sugges ed ha pa hogens could a ge and inhibi cell wall epai ing enzymes such as XET o achie e coloniza ion o he hos issue [ 72 ]. On he o he hand, pec ines e ases, whose deg ee o me hyles e i ica ion de e mines cell wall solidi y and coope a es o i s disassembly [ 73 ], we e down- egula ed in P. pinea, wi h hei inhibi o s PMEI being he mos induced genes in his species a e F. ci cina um in ec ion. In con as , one PMEI was up- egula ed, bu ano he one was down- egula ed in P. adia a ha would esul in inc eased exposu e o pa hogen in ec ion in his species. PMEI ac i i y has been associa ed wi h esis an geno ypes in se e al plan species [ 73 ], and ou esul s indica e i s ole also in he esis ance o F. ci cina um. Ano he s uc u al de ense induced in coni e s is ea ly ibe s ligni ica ion o a es hyphal pene a ion a he si e o in ec ion. P e ious s udies o plan s challenged wi h pa hogens ha e shown he induc ion o genes in ol ed in lignin biosyn hesis [ 29 – 31 , 37 ]. In P. pinea, he main enzymes in ol ed in he lignin biosyn hesis we e induced by F. ci cina um in ec ion, including wo di igen -like disease esis ance p edic ed p o eins ha a e associa ed wi h lignin biosyn hesis and esponse o pa hogens [ 74 ]. No ewo hy, he gene ha encodes he phenylalanine ammonia-lyase (PAL), he enzyme ha leads o lignin biosyn hesis, was no p esen in he DEGs o P. adia a in spi e o i s key ole in linking plan p ima y and seconda y me abolism. These esul s sugges a quick P. pinea esponse In . J. Mol. Sci. 2021,22, 5231 17 o 27 in he ein o cemen o cell walls by ligni ica ion, con as ing wi h he weak egula ion o hese genes in P. adia a. Besides b anching in o lignin, he phenylp opanoid pa hway leads o la onoid /iso la onoid biosyn hesis [ 75 ]. S a ing wi h he up- egula ion o he gene encoding PAL enzyme, he esis an species induced an ac i e and wide de ense eac ion co e ing almos comple ely he la onoid pa hway (Figu e 7), which includes he induc ion o a la ge numbe o CHS1 ( he i s enzyme o his pa hway) and CHI (isome ize chalcone o la anone) [ 76 ] ansc ip s. Recen s udies ha e epo ed an impo an ole o la onoid pa hway componen s in he esis ance o Picea abies o He e obasidion in ec ion [ 37 , 77 , 78 ], being one o he main induced pa hways in asymp oma ic ees. The mode a e esis an P. pinas e challenged wi h F. ci cina um showed ha he induc ion o la onoid biosyn hesis was main ained o e ime (un il 10 dpi) [ 42 ]. In acco dance, al hough chalcone syn hesis was highly induced in P. adia a as well, he downs eam componen s o his pa hway we e poo ly ep esen ed. As la onoids ha e an ioxidan p ope ies [ 79 ], he oxida i e s ess and he subsequen induc ion o SA caused by he F. ci cina um in ec ion could be a enua ed by he ea ly esponse o la onoid compounds and GSH/GST p o eins in P. pinea. In line wi h his, he mos o e exp essed GO e ms and KEGG pa hways in P. pinea we e ela ed o phenylp opanoid, chalcone, la onoid, an hocyanin, and seconda y me aboli e syn hesis. No ewo hy, bo h species showed induc ion o LAR p o eins upon pa hogen in ec ion, which sugges s he p oduc ion o p oan hocyanidins as a esponse o F. ci cina um in ec ion and alkaloid be be ine p o eins ha inhibi he mul iplica ion o ungi [80]. Unde s ess, PR p o eins a e indispensable o plan immune esponses [ 81 ], con- e ing local o sys emic esis ance. In addi ion o PR1 p o eins, a la ge numbe o genes wi h an imic obial p ope ies ha encode o he PR p o eins, including PR2, PR3, PR5, PR9, PR10, and PR14, we e mainly up- egula ed. This is in acco dance wi h se e al s udies on o es ee-pa hogen in e ac ion [ 19 , 27 , 29 , 30 , 32 , 36 , 38 , 40 , 42 , 82 ]. I has been epo ed ha , a e F. ci cina um in ec ion, JA and SA induce chi inases (PR3) [ 83 ] ha deg ade chi in, a majo componen o ungal cell walls. The p esence o β -1,3-endoglucanases (PR2) g ea ly enhances he an i ungal p ope ies o PR3 by deg ading he glucan ma ix in which chi in is embedded [ 84 ]. Se e al genes encoding PR3 p o eins we e up- egula ed in bo h pine species; howe e , a PR2 p o ein was only induced in P. pinea, sugges ing a less e ec i e esponse o P. adia a a an ea ly s age o he disease. Likewise, he plan PR5 amily, known as hauma in-like p o ein (TLP), has also shown an i ungal p ope ies agains se e al o es ee pa hogens [ 27 , 29 , 37 , 54 , 85 ]. Addi ionally, Ca asco e al. [ 38 ] sugges ed he possible ole o PR1 and PR5 in he ac i a ion o he SA-dependen pa hway, esul ing in he induc ion o sys emic induced esis ance (SIR) agains PPC in P. adia a. O he PR p o eins such as pe oxidases (PR9) we e up- and down- egula ed in P. pinea, simila o he in e ac ion o P. pinas e -F. ci cina um a 10 dpi [ 42 ] and Eucalyp us ni ens-Phy oph ho a cinnamomi a 5 dpi [ 36 ]. This could poin ou pine PR9 as e ec o a ge o F. ci cina um, which has been ound in se e al Phy oph ho a species in ec ing Ca ica papaya [ 86 – 88 ]. I has been sugges ed ha ac i a ion o PR genes, no ably plan chi inases, is p oduced mainly in suscep ible Pinus spp. du ing PPC disease [ 19 , 83 , 89 , 90 ]. Howe e , hese s udies ha e ema ked ha in esis an plan s, he highes exp ession le els o hese genes a e de ec ed in ea ly s ages (2–3 dpi), dec easing o e ime, while he opposi e occu s in suscep ible plan s whe e an accumula ion o hese ansc ip s can be no ed a 8–14 dpi. Ou esul s suppo he p e ious conclusions since P. pinea has shown a highe numbe o induced PR genes han P. adia a a 4 dpi. Among coni e s, one o he mos common induced de enses agains pa hogens is an ea ly ligni ica ion o ibe s [ 91 ]. In he up- egula ed genes o he pa hogen when in ec ing P. pinea, he mos ep esen a i e GO e ms we e ela ed o he deg ada ion o he plan cell wall. Indeed, F. ci cina um ansc ip s ma ching genes ha encode o cell wall-deg ading enzymes (CWDE) we e de ec ed in g ea e numbe s in he up- egula ed da ase . Se e al glycoside hyd olases we e iden i ied, which a e esponsible o he hyd olysis o he suga esidues link in cellulose and hemicelluloses [ 92 ]. Among hese, a gene in ol ed in he In . J. Mol. Sci. 2021,22, 5231 18 o 27 con e sion o a plan cell wall polysaccha ides in o e men able suga s, he a abinan endo- 1,5-alpha-L-a abinosidase, was also de ec ed. In addi ion, genes wi h a p edic ed ole in lignin deg ada ion, such as laccases, GMC oxido educ ases, and cellobiose dehyd ogenases, we e uniquely p esen in he up- egula ed gene g oup. This could sugges a di e en ial ac i i y o F. ci cina um in he deg ada ion o lignin be ween P. pinea and P. adia a, which could be associa ed wi h he lignin con en in each species. Mo eo e , he s ong eac ion in cell wall ein o cemen obse ed in P. pinea may be behind he ac i a ion o se e al CWDE by he pa hogen. Addi ionally, se e al e ms ela ed o o ni hine me abolism we e en iched in he up- egula ed genes. O ni hine is a non-p o ein amino acid ha pa icipa es in he plan esponse o s ess, which could indica e seques a ion o po en ial plan de enses o he nu i ional bene i o he pa hogen du ing in asion. On he o he hand, enzymes such as he expansin-like p o ein ha induces ex ensibili y and s ess elaxa ion o plan cell walls [ 93 ] we e iden i ied as down- egula ed in he pa hogen in ec ing P. pinea in compa ison o P. adia a. In addi ion, ungal chi inases ha a e p esumably in ol ed in ungal cell wall emodelings, such as spo e ge mina ion o hyphal ip g ow h [ 94 ], we e also iden i ied in his da ase . The i s s a egy o pa hogens o ob ain nu ien s om he plan in ol es he use o anspo e s, and hei abili y o use hose nu ien esou ces de e mines as much he success o he in asion [ 95 , 96 ]. The unc ional analysis e ealed a no iceable en ichmen o nu ien anspo in he P. pinea down- egula ed genes, especially o ni ogen sou ces including ammonium and amino acids. Genes such as amino acid pe meases (e.g., cholin pe mease and ca ni ine anspo e ), alcohol dehyd ogenases (s-(hyd oxyme hyl) glu- a hione dehyd ogenase alcohol dehyd ogenase), o o mamidases we e p esen in his g oup, being he e o e o e - ep esen ed when in ec ing P. adia a. Fu he mo e, choline anspo , known as he p omo e o Fusa ium g aminea um g ow h and i ulence [ 97 ], was highly down- egula ed in P. pinea in compa ison o P. adia a. In e es ingly, se e al nodulin-like p o eins we e highly induced in P. adia a. The induc ion o hese p o eins in plan -mic obe in e ac ion has been associa ed wi h enhancing pa hogen i ness by i s con ol o e plan anspo e s [ 98 ]. The la ge-scale induc ion o anspo e s o he up ake o nu ien s has been associa ed wi h he shi om he bio ophic o nec o ophic phase in hemibio ophic pa hogens due o he apid g ow h o seconda y hyphae in he la e phase [ 99 ]. Likewise, genes encoding p o eins wi h oles in nu ien anspo we e highly up- egula ed a he ea ly s ages o F. oxyspo um coloniza ion o suscep ible seedlings o Medicago unca ula [ 100 ]. I has been demons a ed ha he ni ogen a ailabili y in ungi modula es i s g ow h, di e en ia ion, and he biosyn hesis o many seconda y me abo- li es [ 101 ]; acco dingly, pa hogens will cause less disease in plan s whe e ni ogen is limi ing [ 102 ]. Mo eo e , a gene al accumula ion o amino acids, concei ably associa ed wi h plan s ess esponse and/o pa hogen hijacking o hos me abolism, was epo ed in he suscep ible P. adia a upon F. ci cina um inocula ion [ 19 ]. The e o e, he en ichmen o genes ela ed o he up ake o ni ogen by F. ci cina um in ec ing P. adia a could p o ide he pa hogen a compe i i e ad an age in he plan -pa hogen in e ac ion. 4. Ma e ials and Me hods 4.1. Fungal Isola e, Plan Ma e ial, and Inocula ion T ial The F. ci cina um isola e (Fc072 ) used in his wo k belongs o ma ing ype 2 (MAT-2) and was isola ed om an in ec ed P. adia a ee loca ed in he No h o Spain (Can ab ia, Spain). Plan ma e ial consis ed o one-yea -old seedlings o P. adia a (P o enance: Galicia, Spain) and P. pinea (P o enance: Mese a No e, Spain). The spo e suspension was ob ained om F. ci cina um cul u ed on PDB medium (2.40% w/ po a o dex ose b o h, Scha lab S.L., Ba celona, Spain). Fo ha , an E lenmeye lask con aining 1 L o PDB and 5 mycelial aga plugs (diame e 4–5 mm) ob ained om he ma gin o an ac i ely g owing colony was placed on an o bi al shake a 140 cycles o 48 h a 25 º C. Finally, he spo e suspension was ob ained by il e ing wice h ough s e ile cheeseclo h o emo e hyphae and was adjus ed wi h a hemocy ome e a 10 6 spo es mL −1 . In . J. Mol. Sci. 2021,22, 5231 19 o 27 Pa hogenici y es s we e ca ied ou by he s em inocula ion echnique [ 7 ]. B ie ly, a wound was made wi h a s e ile scalpel 5–7 cm abo e he colla o each plan a e he emo al o needles om ha a ea. Then 24 seedlings o each species in ull g ow h we e inocula ed wi h 10 µ L o he spo e suspension, and ano he 24 con ol seedlings we e mock-inocula ed in he same way wi h s e ilized dis illed wa e . The inocula ed wound was immedia ely sealed wi h Pa a ilm® o p e en d ying, and he seedlings we e placed in a g ow h chambe a 21.5 ◦ C, wi h a 16/8 h ligh /da k pho ope iod. Wa e ing and o he p ocedu es we e conduc ed as pe ou ine nu se y p ac ice, excep ha no e ilize s o ungicides we e applied. Each biological eplica e was ep esen ed by an indi idual seedling in his s udy. Six biological eplica es pe ea men we e used o physiological pa ame e s, excep o he condi ion o P. pinea inocula ed by F. ci cina um wi h ou biological eplica es. On he o he hand, ou biological eplica es pe ea men we e used o he gene exp ession analysis. Sampling o ansc ip omic analysis and physiological measu emen s occu ed 4 days a e inocula ion (dpi). The emaining seedlings we e used o check he suscep ibili y o each species o F. ci cina um. In pa icula , seedling mo ali y was es ima ed wice a week, and su i al analysis based on he nonpa ame ic es ima o Kaplan–Meie [ 103 ] was pe o med wi h he “Su i al” package [ 104 ] o es he mo ali y up o he end o he expe imen (84 days). Su i al cu es we e c ea ed wi h he “Su i ” unc ion, and he di e ences be ween he cu es we e es ed wi h he “Su di ” unc ion. All analyses we e pe o med using R so wa e en i onmen [105]. 4.2. Wa e Po en ial and Needle Gas Exchange-Rela ed Pa ame e s Midday s em wa e po en ial ( Ψmd , MPa) was measu ed o e e y seedling using a Scholande - ype p essu e chambe (PMS Ins umen Co., Albany, OR, USA). The apical shoo ne CO 2 assimila ion a e (A, µ mol CO2 m −2· s −1 ), anspi a ion a e (E, mmol H 2 O m −2· s −1 ), s oma al conduc ance (gs, mol H 2 O m −2· s −1 ) and sub-s oma al CO 2 concen a- ion (Ci, pm) we e measu ed using an in a- ed gas exchange analyze (LCp o-SD, ADC BioScien i ic Limi ed, Hoddesdon, U.K.) wi h a coni e - ype chambe . The Shapi o–Wilk’s and Ba le ’s es s we e used o es o no mali y and ho- moscedas ici y o he da a (p ≤ 0.05). A wo-way analysis o a iance (ANOVA) was pe o med on each physiological pa ame e in o de o e alua e he F. ci cina um and species e ec . When da a did no ollow ANOVA’s assump ions, obus s a is ical me hods we e applied [ 106 ]. Pa icula ly, he e oscedas ic wo-way ANOVAs we e ca ied ou using he gene alized Welch p ocedu e and a 0.1 immed mean ans o ma ion. ANOVAs we e ca ied ou using he “Wilcox” Robus S a is ics (WRS2)” package, wi h he unc ions “ 3way” and “lincon” [ 107 ] using R so wa e [ 107 ]. Da a a e p esen ed as mean ± SE (s anda d e o ). 4.3. RNA Ex ac ion, Lib a y P epa a ion, and Sequencing To al RNA ex ac ions we e pe o med om 100 mg o s em a he inocula ion poin o each sample using he me hod desc ibed by Valledo e al. [ 108 ]. The o al RNAs we e submi ed o Mac ogen (Mac ogen, Ko ea) o sequencing. Pai ed-end lib a ies wi h agmen s o 150 bp we e p epa ed using poly(A) selec ion wi h he T uSeq S anded mRNA LT sample p epa a ion Ki (Illumina, San Diego, CA, USA) o a co e age dep h o 80 M eads. Subsequen ly, lib a ies we e sequenced by Illumina No aSeq 6000 pla o m. 4.4. P e-P ocessing o Raw Da a and Mapping o Reads Raw eads ha e been deposi ed in he NCBI SRA Da abase unde accession numbe s SRR13737940-53 (BioP ojec PRJNA702546). All RNA sequence iles we e i s assessed o quali y con ol using Fas QC .0.11.9 [ 109 ]. The aw eads we e immed o Illumina adap o sequences and low-quali y base-calls using T immoma ic .0.38 [110]. To pe o m he e e ence-based alignmen o he hos , bo h pine species (P. adia a and P. pinea) we e ea ed equally using he assembled Pinus aeda genome (Pi a_ 2.01; T eegenes In . J. Mol. Sci. 2021,22, 5231 20 o 27 da abase [ 111 ]). Reads we e mapped o he P. aeda genome wi h HiSa 2 .2.0.0 [ 112 ] wi h he de aul se ings o ob ain SAM iles. The iles con aining in o ma ion o he mapped pine eads we e hen analyzed wi h Fea u eCoun s .1.4.0 (Sub ead package) [ 113 ] o ob ain ead coun s o gene egions speci ied in a co esponding GTF ile (Pi a_ 2.01; T eegenes da abase [ 111 ]) o he P. aeda genome sequence. The esul ing coun iles we e hen manually o ma ed in o a coun ma ix sui able o di e en ial exp ession (DE) analysis. Fo he pa hogen, he sequenced genome o he isola e Fc072 o F. ci cina um was used o alignmen wi h HiSa 2 .2.0.0. wi h he de aul se ings o ob ain SAM iles. Due o he lack o a ailable anno a ion iles o his pa hogenic ungus, a ansc ip ome assembly was conduc ed. Be o e assembly, e e y SAM ile was piped o SAM ools u ili y o gene a ing an alignmen ile in bina y alignmen map (BAM) o ma and so ing [ 114 ]. S ingTie .2.1.4 was hen used o assemble he ansc ip ome using he BAM ile om each sample, me ging all assemblies in o an expe imen -le el e e ence assembly and es ima ing he abundances o all ansc ip s assembled, mapping again he eads o he expe imen -le el e e ence [ 115 ]. The ou pu ile was e o ma ed o u he analysis in edgeR using he “p epDE.py” sc ip a ailable a h ps://ccb.jhu.edu/so wa e/s ing ie/index.sh ml? =manual (accessed on 15 Ap il 2020). 4.5. Anno a ion The F. ci cina um expe imen -le el e e ence assembly was con e ed o as a o ma using G ead .0.12.1 [ 116 ]. The ou pu ile was anno a ed wi h EnTAP .0.9.2 [ 117 ] as ol- lows. The pipeline s a ed using GeneMa kS-T .5.1 [ 118 ] o open eading ame p edic ion. A e ha , he simila i y sea ch was conduc ed by BLASTp using he NBCI non- edundan p o ein da abase ( elease-201), Re Seq comple e p o ein da abase ( elease-201), and he UniP o KB/Swissp o da abase ( elease-2020_05) h ough DIAMOND .1.9.2 [ 119 ] wi h de aul se ings. The o hologous g oup assignmen o gene amilies including p o ein domains (SMART/P am), gene on ology (GO) e ms, and KEGG pa hway was pe o med wi h EggNOG .1.0.3 [ 120 ] and In e P oScan .5.47–82.0 [ 121 ]. The p edic ed F. ci cina um p o eins we e also used o in es iga e whe he hey had been e i ied o be pa hogenic genes using anno a ions om he Pa hogen–Hos In e ac ion da abase (PHI-base) [ 122 ] us- ing BLASTp (e- alue < 0.00001). In addi ion, he machine lea ning p edic o E ec o P [ 123 ] ained o ungal sec e ed p o eins we e used o iden i y po en ial e ec o s among he di e en ially exp essed genes o F. ci cina um. The unc ional analysis o he hos ansc ip s was pe o med used he upda ed anno a ion ile o P. aeda (Pi a.2_01.en ap_anno a ions. s ; T eegenes da abase [111]). The ile was manually adjus ed o u he analysis. 4.6. T ansc ip Exp ession Analysis The coun s ma ix ables we e loaded in o he so wa e R .3.6.2 [ 105 ], and bo h pine and ungal RNA-Seq da a we e analyzed using edgeR .1.3.959 package [ 124 ], pe o ming he ollowing s eps. Fi s , da a se s we e no malized in o de o emo e non-biological a ia ion and o make alues compa able ac oss he samples. No maliza ion o ead coun s was conduc ed wi h he immed mean o M- alues (TMM) me hod o edgeR. A e wa d, each ansc ip was i ed o a gene alized linea model ollowing a nega i e binomial dis ibu ion, and s a is ical es ing o he di e en ial gene exp ession (DGE) signi icance was pe o med using he empi ical Bayes quasi-likelihood F- es s. Compu ed p- alues we e adjus ed using he alse disco e y a e (FDR) o Benjamini-Hochbe g o con ol o mul iple es ing [ 125 ]. Pai wise compa isons o he DE o pine ansc ip s we e pe o med o inocula ed s. mock-inocula ed. Fo F. ci cina um ansc ip s, a pai - wise compa ison be ween P. pinea s. P. adia a condi ions was conduc ed. The iden i- ica ion o di e en ial exp ession genes (DEGs) was de e mined using he h eshold o log 2 (|Fold-change|) ≥ 1.5 a a alse disco e y a e o (FDR) lowe han 0.05. To isualize he simila i y o he eplica es and iden i y any sample ou lie s, he p incipal compo- nen analysis (PCA) was pe o med using he log 2 old changes o he hos and he pa hogen da ase s. In . J. Mol. Sci. 2021,22, 5231 21 o 27 4.7. Func ional Analysis In o de o pe o m an e icien unc ional analysis, he DEGs o each da ase we e di ided in o up- and down- egula ed subse s [ 126 ]. Using all genes as backg ound, GO and KEGG en ichmen analysis o he DEGs we e implemen ed by GOSeq .1.38.0 based on he Wallenius non-cen al hype -geome ic dis ibu ion ha allows he adjus men o DEGs leng h bias [ 127 ]. These analyses we e ca ied ou o he h ee di e en compa isons (inocula ed s. mock-inocula ed o bo h pine species and pa hogen gene exp ession compa isons). GO e ms we e conside ed signi ican ly en iched i he p- alue was lowe han 0.05. 5. Conclusions In his a icle, a comp ehensi e ansc ip ional s udy o he hos -pa hogen in e ac ion be ween he hemibio ophic pa hogen F. ci cina um and a esis an and a suscep ible hos species is p esen ed. The lack o a subs an ial esponse in P. adia a con as ed wi h an ad anced ansc ip ional ep og amming o de ense in P. pinea a an ea ly s age o he disease. The esul s ha e sugges ed ha he weak esponse o P. adia a could be ela ed o he impai ed pe cep ion o he ungal in ec ion since ea ly de ense esponses, including calcium lux, ecogni ion by R p o eins, o he ac i a ion o mi ogen-ac i a ed p o ein kinases (MAPKs), we e absen in his species. The ailu e du ing he ini ial in ec ion s age ha in u n a e in ol ed in signaling he in e media e and la e esponses has p esumably led o a weake ac i a ion o a di e se a ay o de ense pa hways, including ligni ica ion, phy oho mone biosyn hesis, and he p oduc ion o non-enzyma ic an ioxidan s such as asco ba e and la onoids. This ci cums ance may enable he ungus o ake ull ad an age o P. adia a nu ien s (such as ni ogen), as sugges ed by he ansc ip s analysis o F. ci cina um. Ou indings allow a be e unde s anding o he pine-F. ci cina um in e ac ion and o coni e de ense esponses o bio ic s ess and se he ounda ion o u u e s udies o alida ing he associa ion o hese candida e genes wi h PPC esis ance ai s. This knowledge will be implemen ed in he b eeding p og ams o he comme cial deploymen o esis an pine ep oduc i e ma e ial. Supplemen a y Ma e ials: The ollowing a e a ailable online a h ps://www.mdpi.com/a icle/10 .3390/ijms22105231/s1, Table S1: Read mapping s a is ics o he hos (Pinus aeda) and he pa hogen (Fusa ium ci cina um) e e ence genomes, Table S2: Lis o di e en ially exp essed P. pinea genes, Table S3: Lis o di e en ially exp essed P. adia a genes, Table S4: Lis o di e en ially exp essed genes o Fusa ium ci cina um, Table S5: Final esul o he EnTAP anno a ion o he Fusa ium ci - cina um ansc ip ome, Table S6: PHI-base alignmen s o he Fusa ium ci cina um ansc ip ome, Table S7 : Signi ican ly en iched GO e ms iden i ied om di e en ially exp essed P. pinea genes, Table S8 : Signi ican ly en iched KEGG pa hways iden i ied om di e en ially exp essed P. pinea genes, Table S9 : Signi ican ly en iched GO e ms iden i ied om di e en ially exp essed P. adia a genes, Table S10 : Signi ican ly en iched KEGG pa hways iden i ied om di e en ially exp essed P. adia a genes, Table S11: Genes in ol ed in plan de ense wi h di e en ial exp ession (log2) in Pinus pinea and P. adia a ollowing challenge wi h Fusa ium ci cina um, Table S12: Phy oho mone ela ed DEGs in he hos s, Table S13: Signi ican ly en iched GO e ms iden i ied om di e en ially exp essed genes in F. ci cina um, Table S14: Fungal ansc ip s ela ed o pa hogenesis exp essed du ing he coloniza ion, Table S15: Alignmen s o he di e en ially exp essed genes o Fusa ium ci cina um o he Machine Lea ning algo i m o he p edic ion o ungal e ec o s, Figu e S1: Wa e po en ial o P. pinea and P. adia a inocula ed wi h F. ci cina um (da k g ay) and con ols (ligh g ay). E o ba s show he s anda d de ia ion. Di e en le e s indica e signi ican di e ences (ANOVA, p< 0.05), Figu e S2: Two-dimensional sca e plo o he p incipal componen analyses (PCA) o Pinus adia a and P. pinea whe e dis ances app oxima e he ypical log2 old changes be ween he samples. Red a ows ep esen indica e he disca ded samples o he downs eam analysis, Figu e S3: Di e en ial gene exp ession was analyzed using he “EdgeR” R package and plo ed as a MA plo . The log2 old change o he no malized gene exp ession be ween inocula ed and con ol ea men s o bo h pine species is ep esen ed on he y-axis, and he a e age log coun s pe million (CPM) a e plo ed on he x-axis. Colo do s ep esen he di e en ially exp essed genes: ed do s indica e In . J. Mol. Sci. 2021,22, 5231 22 o 27 up- egula ed genes, and blue do s down- egula ed genes. Black small do s ep esen genes wi h a simila exp ession be ween ea men s, Figu e S4: Di e en ial gene exp ession was analyzed using he “EdgeR” R package and plo ed as a MA plo . The log2 old change o he no malized gene exp ession o Fusa ium ci cina um in ec ing Pinus pinea in compa ison wi h P. adia a is ep esen ed on he y-axis, and he a e age log coun s pe million (CPM) is plo ed on he x-axis. Colo do s ep esen he di e en ially exp essed genes: ed do s indica e up- egula ed genes, and blue do s down- egula ed genes. Black small do s ep esen ungal genes wi h a simila exp ession be ween pine species. Au ho Con ibu ions: J.M.-G. pe o med he expe imen al wo k. G.P. and J.A. ca ied ou he physiological analysis. C.Z.-B. conduc ed he bioin o ma ics analysis and in e p e ed da a analyses. G.P., A.A., and J.J.D. ob ained unding o suppo he esea ch. L.V. p o ided echnical aspec s o R.N.A.-sequencing and assis ed wi h he c i ical e alua ion o he manusc ip . C.Z.-B. w o e he manusc ip wi h inpu om J.M.-G., J.A., G.P., A.A., and J.J.D., who supe ised aspec s o his esea ch. All au ho s ha e ead and ag eed o he published e sion o he manusc ip . Funding: This esea ch was suppo ed by FEDER h ough COMPETE (P og ama Ope acional Fa o es de Compe i i idade) (POCI-01-FEDER-016785) and by na ional unds h ough he Po - uguese Founda ion o Science and Technology (FCT) wi hin he URGENTpine p ojec (PTDC/ AGR-FOR/2768/2014). Thanks a e due o FCT/MCTES o inancial suppo o CESAM (UID/50017/ 2020+UIDB/50017/2020) h ough na ional unds. FCT also suppo ed J.A. (SFRH/BD/120967/2016) and JM-G (SFRH/BPD/122928/2016). This s udy was also made possible h ough he p ojec PID2019-110459RB-I00 unded by MICINN (Spain) and FEDER (EU) budge as well as he p ojec VA208P20 unded by JCYL (Spain). Ins i u ional Re iew Boa d S a emen : No applicable. In o med Consen S a emen : No applicable. Da a A ailabili y S a emen : T ansc ip ome da a a e a ailable a NCBI da abase (PRJNA702546). Acknowledgmen s: The au ho s hank Ma cos Ga cía o in o ma ics suppo , A oa Suá ez o bioin o ma ics ad ice as well as Se gio Díez o helping in R sc ip ing. We also wan o hank F ancisco J. Colina o echnical assis ance in labo a o y wo k. The da a analysis has been ca ied ou using he esou ces o he “Cen o de Supe compu ación de Cas illa y León” (SCAYLE) unde he aluable echnical suppo o Ca men Cal o and Jesús Lo enzana. Con lic s o In e es : The au ho s decla e no con lic o in e es . Re e ences 1. Mead, D.J. Sus ainable Managemen o Pinus adia a Plan a ions; FAO: Rome, I aly, 2013. 2. MITECO. Anua io de Es adís ica Fo es al 2018. Mad id. 2020. A ailable online: h ps://www.mapa.gob.es/es/desa ollo- u al/ es adis icas/ o es al_anua io_2018.aspx (accessed on 11 No embe 2020). 3. O íz de U bina, E.; Mesanza, N.; A agonés, A.; Raposo, R.; El i a-Recuenco, M.; Boqué, R.; Pa en, C.; Ai ken, J.; I u i xa, E. Eme ging Needle Bligh Diseases in A lan ic Pinus Ecosys ems o Spain. Fo es s 2016,8, 18. [C ossRe ] 4. Bezos, D.; Ma ínez-Al a ez, P.; Fe nández, M.; Diez, J.J. Epidemiology and managemen o pine pi ch canke disease in Eu ope-A e iew. Bal . Fo . 2017,23, 279–293. 5. Wing ield, M.J.; Hamme bache , A.; Ganley, R.J.; S eenkamp, E.T.; Go don, T.R.; Wing ield, B.D.; Cou inho, T.A. Pi ch canke caused by Fusa ium ci cina um-A g owing h ea o pine plan a ions and o es s wo ldwide. Aus alas. Plan Pa hol. 2008 ,37, 319–334. [C ossRe ] 6. Ma ínez-Ál a ez, P.; Pando, V.; Diez, J.J. Al e na i e species o eplace Mon e ey pine plan a ions a ec ed by pi ch canke caused by Fusa ium ci cina um in no he n Spain. Plan Pa hol. 2014,63, 1086–1094. [C ossRe ] 7. Ma ín-Ga cía, J.; Pa aschi , M.; Flo es-Pacheco, J.A.; Chi a, D.; Diez, J.J.; Fe nández, M. Suscep ibili y o Se e al No heas e n Coni e s o Fusa ium ci cina um and S a egies o Biocon ol. Fo es s 2017,8, 318. [C ossRe ] 8. Mulle , M.; Pé ez-Sie a, A.; A mengol, J.; Be begal, M. Pheno ypical and molecula cha ac e isa ion o Fusa ium ci cina um: Co ela ion wi h i ulence and ungicide sensi i i y. Fo es s 2017,8, 458. [C ossRe ] 9. Eschen, R.; B i on, K.; B ocke ho , E.; Bu gess, T.; Dalley, V.; Epanchin-Niell, R.S.; Gup a, K.; Ha dy, G.; Huang, Y.; Kenis, M.; e al. In e na ional a ia ion in phy osani a y legisla ion and egula ions go e ning impo a ion o plan s o plan ing. En i on. Sci. Policy 2015,51, 228–237. [C ossRe ] 10. Ve aino, A.; Po ing, R.; Raposo, R. EU Legisla ion on Fo es Plan Heal h: An O e iew wi h a Focus on Fusa ium ci cina um. Fo es s 2018,9, 568. [C ossRe ] In . J. Mol. Sci. 2021,22, 5231 23 o 27 11. Zamo a-Balles e os, C.; Diez, J.J.; Ma ín-Ga cía, J.; Wi zell, J.; Solla, A.; Ahumada, R.; Cap e i, P.; Clea y, M.; D enkhan, R.; D oˇ ák, M.; e al. Pine Pi ch Canke (PPC): Pa hways o Pa hogen Sp ead and P e en i e Measu es. Fo es s 2019 , 10, 1158. [C ossRe ] 12. Swe , C.L.; Po e , B.; Fou ie, G.; S eenkamp, E.T.; Go don, T.R.; Wing ield, M.J. Associa ion o he pi ch canke pa hogen Fusa ium ci cina um wi h g ass hos s in comme cial pine p oduc ion a eas o Sou h A ica. Sou h. Fo . 2014 ,76, 161–166. [C ossRe ] 13. He nández-Esc ibano, L.; I u i xa, E.; El i a-Recuenco, M.; Be begal, M.; Campos, J.A.; Renobales, G.; Ga cía, I.; Raposo, R. He baceous plan s in he unde s o y o a pi ch canke -a ec ed Pinus adia a plan a ion a e endophy ically in ec ed wi h Fusa ium ci cina um.Fungal Ecol. 2018,32, 65–71. [C ossRe ] 14. Go don, T.R.; Swe , C.L.; Wing ield, M.J. Managemen o Fusa ium diseases a ec ing coni e s. C op P o . 2015 , 73, 28–39. [C ossRe ] 15. Dwinell, D. Global Dis ibu ion o he Pi ch Canke Fungus. In P oceedings o he P oceedings o he IMPACT Mon e ey Wo kshop; De ey, M., Ma heson, A., Go don, T., Eds.; CSIRO Fo es y and Fo es P oduc s, Kings on AC: Mon e ey, CA, USA, 1999; pp. 54–57. 16. Ma ín-Ga cía, J.; Lukace ico a, A.; Flo es-Pacheco, J.A.; Ja ie Diez, J.; D o ak, M. E alua ion o he Suscep ibili y o Se e al Czech Coni e P o enances o Fusa ium ci cina um.Fo es s 2018,9, 72. [C ossRe ] 17. Ma ín-Ga cía, J.; Zas, R.; Solla, A.; Woodwa d, S.; Han ula, J.; Vainio, E.J.; Mulle , M.; Mo ales-Rod íguez, C.; Vannini, A.; Ma ínez-Ál a ez, P.; e al. En i onmen ally- iendly me hods o con olling pine pi ch canke . Plan Pa hol. 2019. [C ossRe ] 18. I u i xa, E.; Ganley, R.J.; Raposo, R.; Ga cía-Se na, I.; Mesanza, N.; Ki kpa ick, S.C.; Go don, T.R. Resis ance le els o Spanish coni e s agains Fusa ium ci cina um and Diplodia pinea. Fo . Pa hol. 2013,43, 488–495. [C ossRe ] 19. Ama al, J.; Co eia, B.; An ónio, C.; Rod igues, A.M.; Gómez-Cadenas, A.; Valledo , L.; Hancock, R.D.; Al es, A.; Pin o, G. Pinus Suscep ibili y o Pi ch Canke T igge s Speci ic Physiological Responses in Symp oma ic Plan s: An In eg a ed App oach. F on . Plan Sci. 2019,10, 509. [C ossRe ] 20. Da ydenko, K.; Nowakowska, J.A.; Kaluski, T.; Gawlak, M.; Sadowska, K.; Ma ín-Ga cía, J.; Diez, J.J.; Oko ski, A.; Oszako, T. A Compa a i e S udy o he Pa hogenici y o Fusa ium ci cina um and o he Fusa ium Species in Polish P o enances o Pinus syl es is L. Fo es s 2018,9, 560. [C ossRe ] 21. Mi chell, R.G.; Wing ield, M.J.; Hodge, G.R.; S eenkamp, E.T.; Cou inho, T.A. The ole ance o Pinus pa ula × Pinus ecunumanii, and o he pine hyb ids, o Fusa ium ci cina um in g eenhouse ials. New Fo . 2013,44, 443–456. [C ossRe ] 22. Roux, J.; Eisenbe g, B.; Kanzle , A.; Nel, A.; Coe zee, V.; Kie zka, E.; Wing ield, M.J. Tes ing o selec ed Sou h A ican Pinus hyb ids and amilies o ole ance o he pi ch canke pa hogen, Fusa ium ci cina um.New Fo . 2007,33, 109–123. [C ossRe ] 23. Kanzle , A.; Nel, A.; Fo d, C. De elopmen and comme cialisa ion o he Pinus pa ula x P. ecunumanii hyb id in esponse o he h ea o Fusa ium ci cina um.New Fo . 2014,45, 417–437. [C ossRe ] 24. Keeling, C.I.; Bohlmann, J. Genes, enzymes and chemicals o e penoid di e si y in he cons i u i e and induced de ence o coni e s agains insec s and pa hogens. New Phy ol. 2006,170, 657–675. [C ossRe ] 25. Vázquez-González, C.; Samped o, L.; Rozas, V.; Zas, R. Clima e d i es in aspeci ic di e en ia ion in he exp ession o g ow h- de ence ade-o s in a long-li ed pine species. Sci. Rep. 2020,10, 10584. [C ossRe ] [PubMed] 26. Rauwane, M.E.; Ogugua, U.V.; Kalu, C.M.; Ledwaba, L.K.; Woldesemaya , A.A.; N ushelo, K. Pa hogenici y and Vi ulence Fac o s o Fusa ium g aminea um Including Fac o s Disco e ed Using Nex Gene a ion Sequencing Technologies and P o eomics. Mic oo ganisms 2020,8, 305. [C ossRe ] [PubMed] 27. Asiegbu, F.O.; Nahalko a, J.; Li, G. Pa hogen-inducible cDNAs om he in e ac ion o he oo o ungus He e obasidion annosum wi h Sco s pine (Pinus syl es is L.). Plan Sci. 2005,168, 365–372. [C ossRe ] 28. Mybu g, H.; Mo se, A.M.; Ame son, H.V.; Kubisiak, T.L.; Hube , D.; Osbo ne, J.A.; Ga cia, S.A.; Nelson, C.D.; Da is, J.M.; Co e , S.F.; e al. Di e en ial gene exp ession in loblolly pine (Pinus aeda L.) challenged wi h he usi o m us ungus, C ona ium que cuum .sp. usi o me. Physiol. Mol. Plan Pa hol. 2006,68, 79–91. [C ossRe ] 29. Adomas, A.; Helle , G.; Li, G.; Olson, A.; Chu, T.; Osbo ne, J.; C aig, D.; Van zyl, L.; Wol inge , R.; Sede o , R.; e al. T ansc ip p o iling o a coni e pa hosys em: Response o Pinus syl es is oo issues o pa hogen (He e obasidion annosum) in asion. T ee Physiol. 2007,27, 1441–1458. [C ossRe ] [PubMed] 30. Azaiez, A.; Boyle, B.; Le ée, V.; Séguin, A. T ansc ip ome P o iling in Hyb id Popla Following In e ac ions wi h Melampso a Rus Fungi. Mol. Plan -Mic obe In e ac . MPMI 2009,22, 190–200. [C ossRe ] 31. Ba aka , A.; Dilo e o, D.S.; Zhang, Y.; Smi h, C.; Baie , K.; Powell, W.A.; Wheele , N.; Sede o , R. Compa ison o he ansc ip omes o Ame ican ches nu (Cas anea den a a) and Chinese ches nu (Cas anea mollissima) in esponse o he ches nu bligh in ec ion. BMC Plan Biol. 2009,9. [C ossRe ] 32. Schlink, K. Iden i ica ion and cha ac e iza ion o di e en ially exp essed genes om Fagus syl a ica oo s a e in ec ion wi h Phy oph ho a ci icola. Plan Cell Rep. 2009,28, 873–882. [C ossRe ] [PubMed] 33. Hayden, K.J.; Ga belo o, M.; Knaus, B.J.; C onn, R.C.; Rai, H.; W igh , J.W. Dual RNA-seq o he plan pa hogen Phy oph ho a amo um and i s anoak hos . T ee Gene . Genomes 2014,10, 489–502. [C ossRe ] 34. Mangwanda, R.; Mybu g, A.A.; Naidoo, S. T ansc ip ome and ho mone p o iling e eals Eucalyp us g andis de ence esponses agains Ch ysopo he aus oa icana.BMC Genomics 2015,16, 1–13. [C ossRe ] [PubMed] 35. B adshaw, R.E.; Guo, Y.; Sim, A.D.; Kabi , M.S.; Che i, P.; Oz u k, I.K.; Hunzike , L.; Ganley, R.J.; Cox, M.P. Genome-wide gene exp ession dynamics o he ungal pa hogen Do his oma sep ospo um h oughou i s in ec ion cycle o he gymnospe m hos Pinus adia a. Mol. Plan Pa hol. 2016,17, 210–224. [C ossRe ] [PubMed] In . J. Mol. Sci. 2021,22, 5231 24 o 27 36. Meye , F.E.; Shuey, L.S.; Naidoo, S.; Mamni, T.; Be ge , D.K.; Mybu g, A.A.; an den Be g, N.; Naidoo, S. Dual RNA-sequencing o Eucalyp us ni ens du ing Phy oph ho a cinnamomi challenge e eals pa hogen and hos ac o s in luencing compa ibili y. F on . Plan Sci. 2016,7, 1–15. [C ossRe ] [PubMed] 37. Ko alchuk, A.; Zeng, Z.; Ghimi e, R.P.; Ki imäenpää, M.; Ra aello, T.; Liu, M.; Muk imin, M.; Kasanen, R.; Sun, H.; Julkunen- Tii o, R.; e al. Dual RNA-seq analysis p o ides new insigh s in o in e ac ions be ween No way sp uce and nec o ophic pa hogen He e obasidion annosum s.l. BMC Plan Biol. 2019,19, 2. [C ossRe ] 38. Ca asco, A.; Weg zyn, J.L.; Du án, R.; Fe nández, M.; Donoso, A.; Rod iguez, V.; Neale, D.; Valenzuela, S. Exp ession p o iling in Pinus adia a in ec ed wi h Fusa ium ci cina um.T ee Gene . Genomes 2017,13. [C ossRe ] 39. Visse , E.A.; Weg zyn, J.L.; S eenkmap, E.T.; Mybu g, A.A.; Naidoo, S. Combined de no o and genome guided assembly and anno a ion o he Pinus pa ula ju enile shoo ansc ip ome. BMC Genom. 2015,16, 1057. [C ossRe ] [PubMed] 40. Visse , E.A.; Weg zyn, J.L.; Mybu g, A.A.; Naidoo, S. De ence ansc ip ome assembly and pa hogenesis ela ed gene amily analysis in Pinus ecunumanii (low ele a ion). BMC Genom. 2018,19, 632. [C ossRe ] 41. Naidoo, S.; Visse , E.A.; Zwa , L.; Du Toi , Y.; Bhadau ia, V.; Shuey, L.S. Dual RNA-seq o Elucida e he Plan -Pa hogen Duel. Cu . Issues Mol. Biol. 2017,27, 127–142. [C ossRe ] 42. He nandez-Esc ibano, L.; Visse , E.A.; I u i xa, E.; Raposo, R.; Naidoo, S. The ansc ip ome o Pinus pinas e unde Fusa ium ci cina um challenge. BMC Genom. 2020,21, 1–18. [C ossRe ] 43. Checke , V.G.; Kushwaha, H.R.; Kuma i, P.; Yada , S. Role o phy oho mones in plan de ense: Signaling and c oss alk. In Molecula Aspec s o Plan -Pa hogen In e ac ion; Singh, A., Singh, I., Eds.; Sp inge : Singapo e, 2018; pp. 159–184. ISBN 9789811073717. 44. Ba i, R.; Jones, J.D.G. Role o plan ho mones in plan de ence esponses. Plan Mol. Biol. 2009,69, 473–488. [C ossRe ] 45. Visse , E.A.; Weg zyn, J.L.; S eenkamp, E.T.; Mybu g, A.A.; Naidoo, S. Dual RNA-seq analysis o he pine-Fusa ium ci cina um in e ac ion in esis an (Pinus ecunumanii) and suscep ible (Pinus pa ula) hos s. Mic oo ganisms 2019,7, 315. [C ossRe ] 46. Ama al, J.; Co eia, B.; Escandón, M.; Jesús, C.; Se ôdio, J.; Valledo , L.; Hancock, R.D.; Dinis, L.-T.; Gómez-Cadenas, A.; Al es, A.; e al. Tempo al physiological esponse o pine o Fusa ium ci cina um in ec ion is dependen on hos suscep ibili y le el: The ole o ABA ca abolism. T ee Physiol. 2020, paa143. [C ossRe ] 47. Zaynab, M.; Fa ima, M.; Abbas, S.; Sha i , Y.; Umai , M.; Za a , M.H.; Bahada , K. Role o seconda y me aboli es in plan de ense agains pa hogens. Mic ob. Pa hog. 2018,124, 198–202. [C ossRe ] 48. Yang, S.F.; Ho man, N.E. E hylene biosyn hesis and i s egula ion in highe plan s. Annu. Re . Plan Biol. 1984 , 35, 155–189. [C ossRe ] 49. De o o, A.; Nie o-Ros o, M.; Xie, D.; Ellis, C.; Ha ms on, R.; Pa ick, E.; Da is, J.; She a , L.; Coleman, M.; Tu ne , J.G. COI1 links jasmona e signalling and e ili y o he SCF ubiqui in-ligase complex in A abidopsis.Plan J. 2002,32, 457–466. [C ossRe ] 50. Liu, H.; Dai, J.; Feng, D.; Liu, B.; Wang, H.; Wang, J. Cha ac e iza ion o a No el Plan ain As Gene, MpAs , ha is Regula ed in Response o In ec ion o Fusa ium oxyspo um .sp. cubense and Abio ic S esses. J. In eg . Plan Biol. 2010 , 52, 315–323. [C ossRe ] [PubMed] 51. Wang, Y.; Wang, Y.; Wang, Y. Apoplas ic P o eases: Powe ul Weapons agains Pa hogen In ec ion in Plan s. Plan Commun. 2020 , 1, 100085. [C ossRe ] [PubMed] 52. Duplessis, S.; Majo , I.; Ma in, F.; Séguin, A. Popla and Pa hogen In e ac ions: Insigh s om Populus Genome-Wide Analyses o Resis ance and De ense Gene Families and Gene Exp ession P o iling. CRC. C i . Re . Plan Sci. 2009,28, 309–334. [C ossRe ] 53. Zhu, Y.; Shao, J.; Zhou, Z.; Da is, R.E. Geno ype-speci ic supp ession o mul iple de ense pa hways in apple oo du ing in ec ion by Py hium ul imum. Ho ic. Res. 2019,6, 10. [C ossRe ] 54. Gaspa , D.; T indade, C.; Usié, U.; Mei eles, B.; Fo es, A.M.; Guima ães, J.B.; Simões, F.; Cos a, R.L.; Ramos, A.M. Compa a i e T ansc ip omic Response o Two Pinus Species o In ec ion wi h he Pine Wood Nema ode Bu saphelenchus xylophilus. Fo es s 2020,11, 204. [C ossRe ] 55. Go , K.E.; Ramonell, K.M. The Role and Regula ion o Recep o -Like Kinases in Plan De ense. Gene Regul. Sys . Bio. 2007 ,1, 167–175. [C ossRe ] 56. Tang, D.; Wang, G.; Zhou, J.M. Recep o kinases in plan -pa hogen in e ac ions: Mo e han pa e n ecogni ion. Plan Cell 2017 ,29, 618–637. [C ossRe ] 57. País, S.M.; Téllez-Iñón, M.T.; Capia i, D.A. Se ine/ h eonine p o ein phospha ases ype 2A and hei oles in s ess signaling. Plan Signal. Beha . 2009,4, 1013–1015. [C ossRe ] 58. Boba, A.; Kos yn, K.; Kozak, B.; Woj asik, W.; P eisne , M.; P escha, A.; Gola, E.M.; Lysh, D.; Dudek, B.; Szopa, J.; e al. Fusa ium oxyspo um in ec ion ac i a es he plas idial b anch o he e penoid biosyn hesis pa hway in lax, leading o inc eased ABA syn hesis. Plan a 2020,251, 50. [C ossRe ] 59. Rod iguez, P.L. Abscisic Acid Ca abolism Gene a es Phaseic Acid, a Molecule Able o Ac i a e a Subse o ABA Recep o s. Mol. Plan 2016,9, 1448–1450. [C ossRe ] 60. Sch oede , J.I.; Namba a, E. A Quick Release Mechanism o Abscisic Acid. Cell 2006,126, 1023–1025. [C ossRe ] [PubMed] 61. Vi as, M.; Ma ín, J.A.; Gil, L.; Solla, A. E alua ing me hyl jasmona e o induc ion o esis ance o Fusa ium oxyspo um,F. ci cina um and Ophios oma no o-ulmi.Fo . Sys . 2012,21, 289–299. [C ossRe ] 62. Fi za, K.N.E.; Payn, K.G.; S eenkamp, E.T.; Mybu g, A.A.; Naidoo, S. Chi osan applica ion imp o es esis ance o Fusa ium ci cina um in Pinus pa ula.Sou h A . J. Bo . 2013,85, 70–78. [C ossRe ] In . J. Mol. Sci. 2021,22, 5231 25 o 27 63. Fäld , J.; Ma in, D.; Mille , B.; Rawa , S.; Bohlmann, J. T auma ic esin de ense in No way sp uce (Picea abies): Me hyl jasmona e- induced e pene syn hase gene exp ession, and cDNA cloning and unc ional cha ac e iza ion o (+)-3-ca ene syn hase. Plan Mol. Biol. 2003,51, 119–133. [C ossRe ] [PubMed] 64. Seo, H.S.; Song, J.T.; Cheong, J.J.; Lee, Y.H.; Lee, Y.W.; Hwang, I.; Lee, J.S.; Choi, Y. Do Jasmonic acid ca boxyl me hyl ans e ase: A key enzyme o jasmona e- egula ed plan esponses. P oc. Na l. Acad. Sci. USA 2001,98, 4788–4793. [C ossRe ] [PubMed] 65. Hi ayama, T.; Kiebe , J.J.; Hi ayama, N.; Kogan, M.; Guzman, P.; Nou izadeh, S.; Alonso, J.M.; Dailey, W.P.; Dancis, A.; Ecke , J.R. RESPONSIVE-TO-ANTAGONIST1, a Menkes/Wilson disease- ela ed coppe anspo e , is equi ed o e hylene signaling in A abidopsis.Cell 1999,97, 383–393. [C ossRe ] 66. Dubois, M.; Ski ycz, A.; Claeys, H.; Maleux, K.; Dhond , S.; De Bod , S.; Vanden Bossche, R.; De Milde, L.; Yoshizumi, T.; Ma sui, M.; e al. ETHYLENE RESPONSE FACTOR6 ac s as a cen al egula o o lea g ow h unde wa e -limi ing condi ions in A abidopsis.Plan Physiol. 2013,162, 319–332. [C ossRe ] 67. Mo a , C.S.; Ingle, R.A.; Wa hugala, D.L.; Saunde s, N.J.; Knigh , H.; Knigh , M.R. ERF5 and ERF6 play edundan oles as posi i e egula o s o JA/E -media ed de ense agains Bo y is cine ea in A abidopsis.PLoS ONE 2012,7, e35995. [C ossRe ] [PubMed] 68. Yang, Y.-X.; Ahammed, G.; Wu, C.; Fan, S.; Zhou, Y.-H. C oss alk among Jasmona e, Salicyla e and E hylene Signaling Pa hways in Plan Disease and Immune Responses. Cu . P o ein Pep . Sci. 2015,16, 450–461. [C ossRe ] 69. Kays, S.J.; Pallas, J.E. Inhibi ion o pho osyn hesis by e hylene. Na u e 1980,285, 51–52. [C ossRe ] 70. He nández-Blanco, C.; Feng, D.X.; Hu, J.; Sánchez-Valle , A.; Deslandes, L.; Llo en e, F.; Be ocal-Lobo, M.; Kelle , H.; Ba le , X.; Sánchez-Rod íguez, C.; e al. Impai men o cellulose syn hases equi ed o A abidopsis seconda y cell wall o ma ion enhances disease esis ance. Plan Cell 2007,19, 890–903. [C ossRe ] [PubMed] 71. Deepak, S.; Shailas ee, S.; Kini, R.K.; Hause, B.; She y, S.H.; Mi hö e , A. Role o hyd oxyp oline- ich glycop o eins in esis ance o pea l mille agains downy mildew pa hogen Scle ospo a g aminicola.Plan a 2007,226, 323–333. [C ossRe ] [PubMed] 72. Muñoz-Be omeu, J.; Lo ences, E.P. Changes in xyloglucan endo ansglucosylase/hyd olase (XTHs) exp ession and XET ac i i y du ing apple ui in ec ion by Penicillium expansum Link. A. Eu . J. Plan Pa hol. 2014,138, 273–282. [C ossRe ] 73. Lione i, V.; Ce one, F.; Bellincampi, D. Me hyl es e i ica ion o pec in plays a ole du ing plan -pa hogen in e ac ions and a ec s plan esis ance o diseases. J. Plan Physiol. 2012,169, 1623–1630. [C ossRe ] 74. Paniagua, C.; Bilko a, A.; Jackson, P.; Dab a olski, S.; Ribe , W.; Didi, V.; House , J.; Gigli-Bisceglia, N.; Wimme o a, M.; Budínská, E.; e al. Di igen p o eins in plan s: Modula ing cell wall me abolism du ing abio ic and bio ic s ess exposu e. J. Exp. Bo . 2017 , 68, 3287–3301. [C ossRe ] 75. Dixon, R.A.; Achnine, L.; Ko a, P.; Liu, C.J.; Reddy, M.S.S.; Wang, L. The phenylp opanoid pa hway and plan de ence-A genomics pe spec i e. Mol. Plan Pa hol. 2002,3, 371–390. [C ossRe ] 76. Dao, T.T.H.; Lin ho s , H.J.M.; Ve poo e, R. Chalcone syn hase and i s unc ions in plan esis ance. Phy ochem. Re . 2011 ,10, 397–412. [C ossRe ] [PubMed] 77. Nemesio-Go iz, M.; Hamme bache , A.; Ih ma k, K.; Källman, T.; Olson, Å.; Lascoux, M.; S enlid, J.; Ge shenzon, J.; El s and, M. Di e en Alleles o a Gene Encoding Leucoan hocyanidin Reduc ase (PaLAR3) In luence Resis ance agains he Fungus He e obasidion pa ipo um in Picea abies.Plan Physiol. 2016,171, 2671–2681. [C ossRe ] [PubMed] 78. Nemesio-Go iz, M.; Blai , P.B.; Dalman, K.; Hamme bache , A.; A ne up, J.; S enlid, J.; Mukh a , S.M.; El s and, M. Iden i ica ion o No way Sp uce MYB-bHLH-WDR T ansc ip ion Fac o Complex Membe s Linked o Regula ion o he Fla onoid Pa hway. F on . Plan Sci. 2017,8, 305. [C ossRe ] 79. Takahama, U.; Oniki, T. Fla onoids and some o he phenolics as subs a es o pe oxidase: Physiological signi icance o he edox eac ions. J. Plan Res. 2000,113, 301–309. [C ossRe ] 80. Schmelle , T.; La z-B üning, B.; Wink, M. Biochemical ac i i ies o be be ine, palma ine and sanguina ine media ing chemical de ence agains mic oo ganisms and he bi o es. Phy ochemis y 1997,44, 257–266. [C ossRe ] 81. Jain, D.; Khu ana, J.P. Role o pa hogenesis- ela ed (PR) p o eins in plan de ense mechanism. In Molecula Aspec s o Plan -Pa hogen In e ac ion; Singh, A., Singh, I., Eds.; Sp inge : Singapo e, 2018; pp. 265–281. ISBN 9789811073717. 82. Gonçal es, M.; Nunes, R.; Tilleman, L.; Van de Pee , Y.; De o ce, D.; Van Nieuwe bu gh, F.; Es e es, A.; Al es, A. Dual RNA Sequencing o Vi is ini e a du ing Lasiodiplodia heob omae In ec ion Un eils Hos –Pa hogen In e ac ions. In . J. Mol. Sci. 2019 , 20, 6083. [C ossRe ] 83. Da is, J.M.; Wu, H.; Cooke, J.E.K.; Reed, J.M.; Luce, K.S.; Michle , C.H. Pa hogen challenge, salicylic acid, and jasmonic acid egula e exp ession o chi inase gene homologs in pine. Mol. Plan -Mic obe In e ac . 2002,15, 380–387. [C ossRe ] [PubMed] 84. Collinge, D.B.; K agh, K.M.; Mikkelsen, J.D.; Nielsen, K.K.; Rasmussen, U.; Vad, K. Plan chi inases. Plan J. 1993 , 3, 31–40. [C ossRe ] 85. Mahomed, W.; an den Be g, N. EST sequencing and gene exp ession p o iling o de ence- ela ed genes om Pe sea ame icana in ec ed wi h Phy oph ho a cinnamomi.BMC Plan Biol. 2011,11, 167. [C ossRe ] 86. Moy, P.; Qu ob, D.; Chapman, B.P.; A kinson, I.; Gijzen, M. Pa e ns o Gene Exp ession Upon In ec ion o Soybean Plan s by Phy oph ho a sojae. Mol. Plan -Mic obe In e ac . 2004,17, 1051–1062. [C ossRe ] [PubMed] 87. Res epo, S.; Mye s, K.L.; Del Pozo, O.; Ma in, G.B.; Ha , A.L.; Buell, C.R.; F y, W.E.; Sma , C.D. Gene p o iling o a compa ible in e ac ion be ween Phy oph ho a in es ans and Solanum ube osum sugges s a ole o ca bonic anhyd ase. Mol. Plan -Mic obe In e ac . 2005,18, 913–922. [C ossRe ] [PubMed]