Phosphorus availability drives mycorrhiza induced resistance in tomato

Phospho us a ailabili y d i es myco hiza induced esis ance in oma o Lau a Dejana 1 , Bea iz Ramı ´ ez-Se ano 2 , Ja ie Ri e o 1 , Jo di Gami 3 , Juan A. Lo ´pez-Ra ´ez 1 and Ma ı ´aJ.Pozo 1 * 1 Depa men o Soil Mic obiology and Symbio ic Sys ems, Es acio ´n Expe imen al del Zaidı ´n, Consejo Supe io de In es igaciones Cien ı ´ficas (CSIC), G anada, Spain, 2 Ins i u de Reche che su la Biologie de l’Insec e (IRBI), UMR 7261, /Uni e si e de Tou s Cen e Na ional de la Reche che Scien ifique (CNRS), Tou s, F ance, 3 Plan Immuni y and Biochemis y G oup, Depa men o Biology Biochemis y and Na u al Sciences, Uni e si a Jaume I, A d. Vicen e Sos Bayna s/n, Cas ello ´n, Spain A buscula myco hizal (AM) symbiosis can p o ide mul iple benefi s o he hos plan , including imp o ed nu i ion and p o ec ion agains bio ic s ess. Myco hiza induced esis ance (MIR) agains pa hogens and insec he bi o es has been epo ed in di e en plan sys ems, bu nu ien a ailabili y may influence he ou come o he in e ac ion. Phospho us (P) is a key nu ien o plan s and insec s, bu also a egula o y ac o o AM es ablishmen and unc ioning. Howe e , li le is known abou how AM symbiosis and P in e ac o egula e plan esis ance o pes s. He e, using he oma o-Funneli o mis mosseae myco hizal sys em, we analyzed he e ec o mode a e di e ences in P e iliza ion on plan and pes pe o mance, and on MIR agains bio ic s esso s including he ungal pa hogen Bo y is cine ea and he insec he bi o e Spodope a exigua. P e iliza ion impac ed plan nu i ional alue, plan de enses, disease de elopmen and ca e pilla su i al, bu hese e ec s we e modula ed by he myco hizal s a us o he plan . Enhanced esis ance o F. mosseae-inocula ed plan s agains B. cine ea and S. exigua depended on P a ailabili y, as no p o ec ion was obse ed unde he mos P-limi ing condi ions. MIR was no di ec ly explained by changes in he plan nu i ional s a us no o basal di e ences in de ense- ela ed phy oho mones. Analysis o ea ly plan de ense esponses o he damage associa ed molecules oligogalac u onides showed p imed ansc ip ional ac i a ion o plan de enses occu ing a in e media e P le els, bu no unde se e e P limi a ion. The esul s show ha P influences myco hizal p iming o plan de enses and he esul ing induced- esis ance is dependen on P a ailabili y, and sugges ha myco hiza fine- unes he plan g ow h s de ense p io i iza ion depending on P a ailabili y. Ou esul s highligh how MIR is con ex dependen , hus un a el molecula mechanism based on plan de ence in will con ibu e o imp o e he e ficacy o myco hizal inoculan s in c op p o ec ion. KEYWORDS DAMPs (damage-associa ed molecula pa e ns), de ense p iming, jasmona e signalling, plan immuni y, plan nu i ion, oligogalac u onides (OGs), he bi o y, pa hogen F on ie s in Plan Science on ie sin.o g01 OPEN ACCESS EDITED BY A emio Mendoza-Mendoza, Lincoln Uni e si y, New Zealand REVIEWED BY Wenwu Zhou, Zhejiang Uni e si y, China Ka in E. G o en, Max Planck Ins i u e o Chemical Ecology, Ge many *CORRESPONDENCE Ma ı ´a J. Pozo [email p o ec ed] SPECIALTY SECTION This a icle was submi ed o Plan Pa hogen In e ac ions, a sec ion o he jou nal F on ie s in Plan Science RECEIVED 03 Oc obe 2022 ACCEPTED 24 No embe 2022 PUBLISHED 19 Decembe 2022 CITATION Dejana L, Ramı ´ ez-Se ano B, Ri e o J, Gami J, Lo ´pez-Ra ´ez JA and Pozo MJ (2022) Phospho us a ailabili y d i es myco hiza induced esis ance in oma o. F on . Plan Sci. 13:1060926. doi: 10.3389/ pls.2022.1060926 COPYRIGHT © 2022 Dejana, Ramı ´ ez-Se ano, Ri e o, Gami , Lo ´pez-Ra ´ez and Pozo. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (CC BY). The use, dis ibu ion o ep oduc ion in o he o ums is pe mi ed, p o ided he o iginal au ho (s) and he copy igh owne (s) a e c edi ed and ha he o iginal publica ion in his jou nal is ci ed, in acco dance wi h accep ed academic p ac ice. No use, dis ibu ion o ep oduc ion is pe mi ed which does no comply wi h hese e ms. TYPE O iginal Resea ch PUBLISHED 19 Decembe 2022 DOI 10.3389/ pls.2022.1060926 In oduc ion The de elopmen o sus ainable echnologies o c op managemen aiming o educe he use o e ilize s and pes icides is an ongoing global challenge in ag icul u e (P e y and Bha ucha, 2018). In his scena io, he po en ial o beneficial symbio ic mic obes o imp o e plan nu i ion and s ess esis ance/ ole ance is well es ablished (Be endsen e al., 2012; Pie e se e al., 2014;Pozo e al, 2020). Howe e , hei pe o mance unde ag onomic condi ions is no op imal, as he ou comes o plan -mic obe in e ac ions a e highly con ex -dependen and may a y acco ding o he plan g owing condi ions ( an de Heyde e al., 2017;Lee Dı  az e al., 2021). Among he beneficial o ganisms used as inoculan s in ag icul u e, ungi, and pa icula ly a buscula myco hizal ungi (AMF), a e ecei ing inc easing in e es (Pozo e al., 2021). These soil-bo ne mic oo ganisms, belonging o he phylum Glome omyco a, a e widesp ead in na u al and ag icul u al ecosys ems and colonize he oo s o mo e han 80% o e es ial plan species, including majo c ops (Smi h and Smi h, 2011). This mu ualis ic associa ion, known as myco hiza, has majo benefi s o bo h pa ne s: AMF ecei e plan o ganic ca bon in he o m o ca bohyd a es and lipids (Keyme e al., 2017;Salme on-San iago e al, 2021). In e u n, AMF imp o e wa e and nu ien s up ake by he plan , especially phospho us (P) (Chen e al., 2018;Fe ol e al, 2019). In addi ion, hey also inc ease plan pheno ypic and me abolic plas ici y o cope wi h bio ic and abio ic s esso s (Ri e o e al., 2018;Sanchez- Bel e al, 2018;Campo e al, 2020;Mi a e al., 2021;Ri e o e al., 2021;Pozo de la Hoz e al., 2021). AM symbiosis usually ende s he plan mo e esis an o ce ain soil-bo ne and abo eg ound pa hogens and chewing he bi o es, as shown in mul iple sys ems (Jung e al., 2012; Song e al., 2013;Song e al., 2015;Sel a aj and Thanga el, 2021; Dowa ah e al., 2022). This Myco hiza-Induced Resis ance (MIR) is ela ed o an imp o ed abili y o myco hizal plan s o igge de ense esponses upon challenge, a cos -e ficien s a egy known as de ense p iming (Pozo and Azcon-Aguila , 2007;Ma inez-Medina e al., 2016;Mauch-Mani e al., 2017). P iming is a common mechanism du ing induced sys emic esis ance igge ed by beneficial mic obes, consis ing o a s onge and as e ac i a ion o plan de ense mechanisms, usually dependen on jasmona e signaling (Pie e se e al., 2014; Mo a-Rome o e al., 2014;G uden e al., 2020). Despi e p iming o plan de enses in esponse o pa hogens and he bi o es in myco hizal plan s is well documen ed (Campos-So iano e al., 2012;Song e al., 2013;Song e al., 2015;Sanchez-Bel e al., 2016; Fio illi e al., 2018;Schoenhe e al., 2019;Sanma ı  n e al., 2020;Ri e o e al., 2021;Man esa-G ao e al., 2022) consequences o AM symbiosis o insec he bi o es a e complex o p edic . Indeed, he nu i ional benefi s o he symbiosis may inc ease ood a ailabili y o imp o e he die quali y o he pes , depending on he ungal and insec iden i y and g owing condi ions (Ko iche a e al., 2009;F ew and P ice, 2019). Thus, he in e ac ion ou come esul s om he in e play o opposi e e ec s: nu i ional imp o emen , benefi ing he he bi o e, and de ense p iming, po en ially educing he bi o e pe o mance (Pozo e al., 2020). Indeed, plan -mic obe-insec in e ac ions a e e y complex, wi h plan s coo dina ing hei esponses acco ding o mul iple ex e nal and in e nal cues h ough a p ecise egula ion o ches a ed by phy oho mone ne wo ks (Pozo e al., 2015;G uden e al., 2020). Many bio ic and abio ic ac o s influence myco hizal coloniza ion (Diagne e al., 2020;Mi a e al., 2021). Indeed, he plan -AMF in e ac ion is finely egula ed by se e al ac o s, including he plan and ungal geno ypes and en i onmen al condi ions, wi h nu ien s a ailabili y as one o he mos influen ial aspec s (Pozo e al., 2015;Lee Dı  az e al., 2021). P is a cen al egula o o myco hizal es ablishmen , oo coloniza ion and ungal g ow h wi hin he hos plan . Thus, he abuse o P e ilize s inhibi s he es ablishmen o myco hizal symbiosis in di e en plan s (Smi h and Smi h, 2011;Smi h e al., 2011;Higo e al., 2020). P, an essen ial nu ien o plan s, is a non- enewable esou ce and poo ly a ailable in he field, being a limi ing ac o o plan g ow h (Wang e al., 2021). This elemen is aken up om he soil in he o m o ino ganic P by he oo s (Rouached e al., 2010), and i s deficiency igge s impo an changes in plan physiology and biochemis y including changes in plan g ow h, me abolism, ho monal balance, gene exp ession and oo a chi ec u e. P deficiency u he p omo es he p oduc ion o he phy oho mones s igolac ones, accumula ion o an hocyanins and o he phenolic compounds (Ha and T an, 2014;Vyso skaya e al., 2016;Ma o e al., 2022). In ag icul u e, chemical and o ganic e ilize s a e s ongly and widely applied o sol e P soil deficiency, causing con amina ion o aqui e s and al e ing plan in e ac ions wi h beneficial mic obes, including AMF (Smi h and Smi h, 2011; Co dell and Whi e, 2015). P le els also impac plan in e ac ions wi h o he o ganisms by al e ing plan issues’nu i ional alue o o ganisms eeding on hem (pa hogens and he bi o es) and by modula ing plan de enses (B euillin e al, 2010;Cas illo e al, 2017;Chan e al., 2021). The ole o P in plan immuni y is cu en ly unde in ense sc u iny, and he da a e eal a e y complex scena io (Chan e al., 2021;Qu e al., 2021;Val- To eg osa e al, 2022). The phospha e s a a ion esponse in plan s has been sugges ed o inc ease plan esis ance o nec o ophic pa hogens and lea chewing he bi o es in se e al plan species (A abidopsis, oma o, obacco) poin ing o a posi i e c oss- alk be ween he JA- and P- s a a ion signaling pa hways ha ac i a es plan immuni y (Khan e al., 2016). Howe e , opposi e esul s ha e been also ound in di e en plan species acing di e en agg esso s (Campo e al., 2020;Val- To eg osa e al, 2022). I should be no ed ha mos basic s udies dealing wi h he molecula mechanisms media ing P e ec s on plan immuni y compa e e y con as ing P le els, usually a om hose used in ag icul u al se ings. Dejana e al. 10.3389/ pls.2022.1060926 F on ie s in Plan Science on ie sin.o g02 Despi e he inc easing numbe o s udies add essing P influence on plan de enses, only a ew ha e in es iga ed he ela ionship be ween P and MIR, sugges ing a complex in e ac ion wi h con as ing esul s (Wang e al., 2020;Qu e al., 2021). While e idence o he ole o P in egula ing myco hizal coloniza ion, plan g ow h and immuni y esponses exis , he in e play among he di e en e ec s and mechanisms behind his in e play emain poo ly s udied (Wang e al., 2020;Qu e al., 2021). Ou s udy aims o elucida e whe he he myco hizal e ec on oma o esis ance o bio ic s esso s is egula ed by P a ailabili y. Fo ha , we e alua ed he impac o di e en P e iliza ion egimes on oma o g ow h, myco hizal coloniza ion and he bi o e esis ance using he oma o/ Funneli o mis mosseae sys em and he nec o ophic pa hogen Bo y is cine ea and he gene alis chewing he bi o e Spodop e a exigua. We ound ha P a ailabili y s ongly influences plan g ow h and esis ance o bio ic s esses, and ha hese e ec s a e modula ed by he AM symbiosis, showing ha MIR is indeed dependen on P a ailabili y. By analyzing plan nu ien s, de ense- ela ed phy oho mones and ansc ip ional egula ion o de enses in esponse o he damage-associa ed signals oligogalac u onides (OGs), we aimed o unco e he mechanis ic basis o he impac o P a ailabili y on MIR. This knowledge will con ibu e o imp o e AMF applica ion and managemen o sus ainable c op p o ec ion. Ma e ial and me hods Biological ma e ial and myco hizal inocula ion The AMF Funneli o mis mosseae BEG12 (Young, 2015) ob ained om he In e na ional Bank o Glome omyco a (h p://www.i-beg.eu), was main ained a he EEZ-CSIC g eenhouse as open-po cul u es o T i olium epens mixed wi h So ghum ulga e plan s using e miculi e-sepioli e subs a e. The inoculum consis ed in he subs a e con aining colonized oo agmen s, ungal mycelia and spo es. Bo y is cine ea was cul i a ed in po a o dex ose aga pla es, supplemen ed wi h eeze-d ied oma o lea es. Th ee weeks la e , B. cine ea spo es we e collec ed om pla es in 0.5X po a o dex ose b o h as p e iously desc ibed (Sanma ı n e al., 2020). Spodop e a exigua (Lepidop e a: Noc uidae) eggs we e p o ided by D . S. He e o lab (ERI-BIOTECMED, Uni e si a de Valencia, Spain). La ae we e ea ed on a ificial die (G eene e al., 1976) a 25°C wi h 16:8h ligh : da k egime and 70% ela i e humidi y un il L2-L3 la al s age, o hei applica ion. Toma o seeds (Solanum lycope sicum L. c . Moneymake ) we e su ace disin ec ed by imme sion in 4% NaHClO (10 min), insed ho oughly wi h s e ile wa e and incuba ed o 10 days in an open con aine wi h s e ile e miculi e a 25°C. Plan le s we e ans e ed o 100 mL po s con aining a s e ile sand: e miculi e (1:1) mix u e. Po s o myco hizal ea men s we e inocula ed by adding 10% ( / ) F. mosseae inoculum. All plan s, including non-inocula ed ones, ecei ed a 3 ml aliquo o a fil a e (<20 mm) o he inoculum, in o de o p o ide he gene al mic obial popula ion bu ee o AMF spo es. Expe imen al design Di e en expe imen s we e ca ied ou o elucida e he e ec o P nu i ion on plan pe o mance and he bi o e esis ance in myco hizal and non-myco hizal plan s. All expe imen s included oma o plan s inocula ed wi h F. mosseae (Fm) o no (Nm). We pe o med a fi s sc eening using h ee P concen a ions: 0.3 mM, 0.6 mM and 1.0 mM (10 plan s pe ea men ). Fo his, plan s we e wa e ed wi h Hewi nu ien solu ion (Hewi , 1996), modified in he P (H 2 NaPO4) concen a ion as desc ibed in Table S1. Plan s we e ha es ed a e 8 weeks o g ow h and he pe o mance o S. exigua ed on de ached lea es was e alua ed (see he bi o e bioassays below). Plan biomass, myco hizal coloniza ion, plan nu ien s, an hocyanin con en and phy oho mone le els we e also de e mined. Based on he esul s ob ained, 0.3 o 0.7 mM P concen a ions we e selec ed o ollow-up expe imen s. In a second expe imen , we assessed P e ec s on Bo y is cine ea lesion de elopmen h ough a de ached lea assay as desc ibed below, and we e alua ed he pe o mance o S. exigua la ae di ec ly ed on Fm and Nm plan s (whole plan bioassay). Six- weeks-old plan s we e in es ed wi h S. exigua la ae and ca e pilla pe o mance (weigh , su i al and pupa ion) was s eadily moni o ed o 3 weeks (9 plan s pe ea men ). Finally, ea ly plan de ense esponses we e compa ed in myco hizal and non-myco hizal plan s g owing a hese wo P le els (0.3 and 0.7 mM; 6 plan s pe ea men ). Fo ha , we assessed he plan esponse o damage by using oligogalac u onides (OGs), well cha ac e ized damage- associa ed molecula pa e ns (DAMPs) (see Plan ea men wi h oligogalac u onides below). The le els o de ense- ela ed phy oho mones and de ense- ela ed gene exp ession we e de e mined 6 hou s pos ea men (hp ) as desc ibed below. Thus, he ac o s conside ed in his s udy we e M: myco hizal inocula ion (le els: Nm, Fm), P: P e iliza ion egimes (le els 0.3, 0.7 and 1.0 mM), and OG: Oligogalac u onide ea men (le els: -OG, +OG). Plan g owing condi ions Fo he di e en expe imen s, plan s we e andomly dis ibu ed and g own in a g eenhouse a 24/16°C wi h a 16/ Dejana e al. 10.3389/ pls.2022.1060926 F on ie s in Plan Science on ie sin.o g03 8 h diu nal pho ope iod and 70% humidi y. Plan s we e wa e ed wice a week wi h hal s eng h Hewi nu ien solu ion (Hewi , 1996)modified in he P con en as desc ibed abo e, and wa e was supplied as needed. Upon ha es ing shoo s and oo s, esh weigh was de e mined and he ma e ial was immedia ely ozen in liquid N and s o ed a -80°C o u he analyses. An aliquo o each indi idual oo sys em was p ese ed o myco hizal quan ifica ion. Bo y is cine ea in ec ion The ou h lea o oma o plan s was de ached o pa hogen bioassays. Pa hogen in ec ion was pe o med by applying 10 mL d ops con aining a conidia suspension (1 × 10 6 spo es/ml) o he de ached lea es. One lea pe plan was inocula ed by adding wo d ops pe leafle , fi e leafle s pe lea , wi h a o al o 90 lesions. The in ec ed lea es we e main ained in 15cm Pe i dishes on we fil e pape and incuba ed in a phy o on chambe a 80% o humidi y and 22°C. Nec o ic lesions we e measu ed 3 days pos -inocula ion. He bi o e pe o mance bioassays Insec pe o mance was e alua ed in wo di e en bioassays, using de ached lea es o whole plan s. Fo he de ached lea es assay, wo second-ins a S. exigua la ae we e placed on one de ached lea o each oma o plan , placed on we fil e pape in 15 cm Pe i dishes. Pla es we e hen incuba ed in a phy o on a 26-24°C, 16:8h day/nigh and 60% ela i e humidi y. La al mo ali y and weigh was e alua ed e e y 2 days o 8 days. Fo whole plan s assay, wo second-ins a la ae we e placed in a leafle o he ou h ue lea o each oma o plan , using clip- cages o a oid hei escape, as desc ibed in Ri e o e al. (2021). E e y wo days, clip-cages we e mo ed o new esh leafle s and ca e pilla biomass, mo ali y and pupa ion we e moni o ed. Plan ea men wi h oligogalac u onides Fo he analysis o ea ly plan esponses, he damage associa ed molecules oligogalac u onides (OGs) we e used o elici plan de ense esponses. The OGs (DP 10-15) we e p epa ed as desc ibed in Benede i e al. (2017) and p o ided by D . De Lo enzo lab (Depa men o Biology and Bio echnology “Cha les Da win”BBCD, La Sapienza Uni e si y, Rome, I aly). Six weeks pos AMF inocula ion oma o plan s g own a 0.3 o 0.7 mM P e iliza ion egimes we e ea ed wi h an aqueous solu ion o OGs (50 mg/ml in milliQ wa e ) as desc ibed in Gami e al. (2020). The ou h ue lea o each plan was sp ayed wi h he OG solu ion o milliQ wa e o con ol plan s using an ae og aph un il unning o . T ea ed lea es we e ha es ed a e 6 hou s o s udy ea ly plan de ense esponses, as his ime was he mos app op ia e o iden i y changes in ho mone con en s and in he exp ession le els o OG esponsi e, de ense ela ed (Gami e al., 2020). De e mina ion o myco hizal coloniza ion AM coloniza ion was measu ed a e clea ing washed oo s in KOH (10%) and s aining ungal s uc u es wi h 5% ink in 2% ace ic acid (Vie heilig e al., 2005). The pe cen age o o al oo leng h colonized by F. mosseae was es ima ed acco ding o he g idline in e sec ion me hod (Gio anne i and Mosse, 1980) using a BOECO zoom s e eo mic oscope Model BST-606. An hocyanin con en An hocyanin con en , as P s a a ion indica o , was e alua ed by adap ing he p o ocol es ablished in Rabino and Mancinelli (1986). F ozen lea es we e g inded and lyophilized, and 5 mg aliquo o d y issue was used pe sample. The pigmen s we e ex ac ed by shaking he plan eeze-d ied lea powde in acidic (1% HCI, w/ ) me hanol in da k o e nigh . Ex ac s we e cen i uged o 10 min a 13000 pm. The amoun o an hocyanin was calcula ed measu ing abso bance a 530 and 657 nm o c ude ex ac , using he o mula A530 - 0.25 A657 o compensa e o he con ibu ion o chlo ophyll and i s deg ada ion p oduc s o he abso p ion a 530 nm. Six independen biological eplica es we e analyzed pe ea men . De e mina ion o mine al nu ien s in lea es Nu ien analyses we e pe o med a he Technical Se ices o he Es acion Expe imen al del Zaidı  n (CSIC). F ozen lea es we e g inded and lyophilized, and 50-100 mg aliquo o d y issue was used pe sample. The concen a ion o P and mic onu ien s we e de e mined a e acid diges ion o samples, by induc i ely coupled plasma op ical emission spec ome y (ICP‐OES; Va ian ICP 720‐ES). To al C and N con en s we e analyzed using an Elemen al Analyze (LECO T uSpec CN), acco ding o s anda d p ocedu es. Six independen biological eplica es we e analyzed pe ea men . Ta ge ed ho monal ex ac ion and quan ifica ion Ho mone ex ac ion was pe o med om eeze-d ied powde ed plan lea es as desc ibed in Sanchez-Bel e al. Dejana e al. 10.3389/ pls.2022.1060926 F on ie s in Plan Science on ie sin.o g04 (2016). Six independen biological eplica es pe ea men we e analyzed. B iefly, 30 mg o plan d y issue was ex ac ed wi h 1mlo H 2 O:MeOH (9:1) con aining 0.001% o HCOOH and 100 ng/ml o in e nal s anda ds. A e di e en cen i uga ions and esuspensions, an aliquo o he ex ac was injec ed in o an Acqui y Ul a Pe o mance Liquid Ch oma og aphy sys em (UPLC) (Wa e s, Mild o d, MA, USA). Ho mones we e ch oma og aphically sepa a ed using an HPLC Kine ex C18 analy ical column (Phenomenex) connec ed o a iple quad upole mass spec ome e (TQD, Wa e s, Manches e , UK). The ch oma og aphic and mass spec ome y condi ions we e hose used by Gami e al. (2012). Ho mone quan ifica ion (ng/g d y weigh ) was pe o med using calib a ion cu es wi h each pu e chemical s anda d. The plan ho mones abscisic acid (ABA), indolace ic acid (IAA), jasmonic acid (JA), i s p ecu so (+)-12-oxo-phy odienoic acid (OPDA) and salicylic acid (SA) we e de e mined. Analysis o gene exp ession by qPCR The exp ession o ma ke genes om di e en me abolic pa hways was analyzed by eal ime quan i a i e PCR (qPCR). Six independen biological eplica es pe ea men we e used. To al RNA om oma o lea es was ex ac ed and ea ed wi h DNase using he Di ec -zol RNA MiniP ep ki (Zymo Resea ch). Subsequen ly, he RNA was pu ified h ough a column using he RNA Clean and Concen a o -5 ki (Zymo Resea ch), and s o ed a -80°C un il use. The fi s -s and cDNA was syn hesized wi h 1 mg o pu ified o al RNA using he iSc ip cDNA Syn hesis ki (Bio-Rad). All ki s we e used acco ding o he manu ac u e ’s sugges ed p o ocols. The exp ession o h ee di e en housekeeping genes, ac in (Solyc03g078400), elonga ion ac o 1‐a(Solyc06g005060) and b‐ ubulin (Solyc04g081490) was measu ed o find he op imal no maliza ion gene, using he No mfinde so wa e (h ps:// moma.dk/no mfinde -so wa e)(Ande sen e al., 2004). Acco ding o he esul s, exp ession alues we e no malized using he housekeeping gene b- ubulin and ela i e quan ifica ion o specific mRNA le els was pe o med using he compa a i e 2–D(DC ) me hod (Li ak and Schmi gen, 2001). The sequences o he specific p ime s used a e shown in Table S2. S a is ical analyses All s a is ical analyses (mul i-way ANOVAs and pos hoc es s applied when app op ia ed, as indica ed in he co esponding figu e legends) we e conduc ed using S a g aphics Plus 3.1 (Rock ille, MD, USA) o ‘R’so wa e .3.5.2 (R De elopmen Co e Team). Figu es we e ob ained using ggplo 2 R package (Wickham e al, 2016). T ea men e ec s on la al su i al we e assessed by compa ing he su i al cu es using he Kaplan-Meie es ima o (Kaplan and Meie , 1958). Su i al dis ibu ion compa ison be ween ea men s was pe o med using he non-pa ame ic Log ank es (Man el-Cox). Su i al analyses we e pe o med using su i al and su mine R packages. Model alida ions we e pe o med using Shapi o-Wilk and Le ene’s es s. Resul s P e iliza ion le els impac myco hizal coloniza ion, plan g ow h and he bi o e pe o mance in oma o To explo e how myco hizal de elopmen and i s e ec s on plan and ca e pilla pe o mance a e a ec ed by P e iliza ion, we compa ed 3 e iliza ion egimes di e ing only in he P con en , anging om limi ing o su ficien P (0.3, 0.7 and 1.0 mM). Analysis o he plan biomass confi med ha P le els had a significan impac on plan g ow h (p<0.001) (Figu es 1A,B). Plan s g own a 0.3 mM P showed abou 50% educed oo and shoo weigh s. Significan di e ences be ween 0.7 and 1.0 mM P we e also obse ed; howe e , hese we e mild compa ed o he mos P limi ing condi ions (Figu es 1A,B). In e es ingly, myco hiza ion did no ha e a global significan e ec on plan esh weigh , bu he e was a significan in e ac ion be ween he P and myco hizal ea men s, wi h myco hiza p omo ing plan g ow h only a he in e media e (0.7 mM) P le el and ep essing oo biomass a he highes P le el (Two way ANOVA, Figu es 1A,B). The e alua ion o an hocyanin accumula ion in lea es, as an indica o o plan P-s a a ion esponse, also confi med he dose-dependen e ec s o P e iliza ion on he plan s. Plan s g own unde low P (0.3 mM) showed he highes an hocyanin le els, while he le els in plan s g owing a 0.7 and 1.0mMwe eno significan ly di e en (Figu e 1C). Myco hiza ion also had a significan e ec educing he an hocyanin le els ( wo-way ANOVA; p<0.05). Myco hizal coloniza ion was also significan ly impac ed by P e iliza ion, wi h inc easing P concen a ions leading o a educ ion in coloniza ion (Figu e 1D). Di e ences we e significan al eady a e 4 weeks o g ow h, wi h myco hizal coloniza ion in he mode a e and high P condi ions being hal o hose a low P. The e ec was mo e p onounced a he la e ime poin (final ha es , 8 weeks), as oo coloniza ion con inued o inc ease in plan s e ilized wi h he lowes P concen a ion (0.3 mM), bu no wi h he o he P le els (Figu e 1D). The e ec o plan P e iliza ion on he pe o mance o S. exigua la ae ed on lea es o hose plan s was also e alua ed by using de ached lea es. La al weigh was influenced by P le els, being significan ly lowe a 0.3 mM P (Figu e 1E). No di e ences be ween la ae ed in 0.7 o 1.0 mM we e ound, ega dless o he myco hizal s a us o he plan (Figu e 1E). P le els also had a Dejana e al. 10.3389/ pls.2022.1060926 F on ie s in Plan Science on ie sin.o g05 B CD EF A FIGURE 1 Impac o P le els on plan g ow h, AM coloniza ion and he bi o e pe o mance. Plan g ow h pa ame e s, ungal coloniza ion and he bi o e pe o mance on non-myco hizal (Nm) and myco hizal oma o plan s colonized by Funneli o mis mosseae (Fm). Plan s we e e ilized wi h di e en P concen a ions: 0.3 mM, 0.7 mM and 1mM o H 2 NaPO₄.(A) Shoo and (B) oo esh weigh (n=10), and (C) an hocyanin con en (n=6) we e de e mined in oma o plan s a ha es , 8 weeks pos myco hizal inocula ion (pmi). (D) Pe cen age o oo leng h colonized by he myco hizal ungi a 4 and 8 weeks pmi (n=10). One lea pe plan was de ached 8 weeks pmi and in es ed wi h 2 second ins a S. exigua la ae (n=20), and (E) weigh o he la ae was de e mined a e 6 days o eeding. (F) La al mo ali y was moni o ed du ing 8 days o con inuous eeding on he de ached oma o lea es. Da a om A o E ep esen means o he n independen biological eplica es ± SD. Two-way ac o ial ANOVA (A–C, E, F) using AM symbiosis (M) and P ea men s (P) as ac o s, o (D) using ime (T) and P ea men s (P) as ac o s we e pe o med, and significance alues o each ac o and hei in e ac ions a e indica ed in he uppe igh co ne o each g aph. As e isks deno e significan e ec o a ac o and hei in e ac ion. ns: no significan ; *: p < 0.5; **:p < 0.01; ***:p < 0.001; ****:p < 0.0001. Di e en le e s ep esen s a is ically significan di e ences (ANOVA, Fishe ’s Leas Significan Di e ence (LSD) es ; p<0.05). Fo (F), da a ep esen he pe cen age o mo ali y a he di e en ime poin s, and he di e ences in he su i al dis ibu ion acco ding o P, M and MxP we e pe o med using he non- pa ame ic Log- ank (Man el-Cox) es . Dejana e al. 10.3389/ pls.2022.1060926 F on ie s in Plan Science on ie sin.o g06 significan impac on S. exigua mo ali y, showing he highes mo ali y a he low 0.3 P le el ( anging be ween 50-60%) (Figu e 1F). A highe P le els (0.7 and 1.0 mM) la ae mo ali y anged om 6 o 21% (Figu e 1F). Al hough highe mo ali y was ound in Fm compa ed o Nm plan s a hese medium and high P le els (21% Fm s 6% Nm o 0.7mM, and 16%Fm s 6%Nm o 1.0 mM), he di e ences we e no significan . No ewo hy, when analyzing he da a sepa a ely acco ding o he myco hizal s a us, P e ec on la al su i al was mo e p onounced in Nm han in Fm plan s: P le els had a e y significan impac on la al su i al when eeding on Nm plan s (p<0.0001), bu no on hose eeding in myco hizal plan s (p=0.052) (Figu e S1A). P e ec s on he bi o e pe o mance depends on he myco hizal s a us o he plan and de e mine MIR O e all, he esul s o he P dose sc eening e ealed ha mos di e ences occu be ween he low (0.3mM) and he highe P egimes (0.7 and 1.0 mM) ha showed simila alues o mos pa ame e s. Acco dingly, 0.3 and 0.7 mM le els we e selec ed o u he expe imen s as he closes doses wi h con as ing e ec s. Bioassays on de ached lea es a e use ul o quick sc eenings o majo e ec s, bu hese e ec s a e usually weake han hose using whole plan bioassays. The la e allow a mo e ealis ic se up and longe e alua ion pe iods. The e o e, we pe o med a second expe imen ocused on he selec ed P le els (0.3 and 0.7 mM) o be e add ess he e ec o myco hiza ion on la al pe o mance unde di e en P e iliza ion. Myco hizal coloniza ion by F. mosseae was 14% and 6% o he 0.3 and 0.7 mM P le els, espec i ely. Again, P e ec on S. exigua mo ali y was significan o la ae eeding in Nm plan s (p<0.0001), bu no o hose eeding on myco hizal (Fm) ones (p=0.27) (Figu e S1B). Thus, myco hizal coloniza ion seems o bu e he s ong e ec o P on plan esis ance o he pes . As in he p e ious expe imen using de ached lea es, la ae pe o med wo s in he lowes P e ilized plan s, showing highe mo ali y le els (Figu es 2A,B), lowe weigh (Figu es 2C,D) and wo s de elopmen –e alua ed as he pe cen age o indi iduals eaching he pupal s age- (Figu es 2E,F). Rega ding he e ec o myco hiza ion on S. exigua pe o mance, unde low P no significan changes we e ound in mo ali y no de elopmen be ween Fm and Nm plan s (Figu es 2A,E), and la al weigh was e en highe a some ime poin s in myco hizal plan s (Figu e 2C). In con as , unde mode a e P le els (0.7 mM), la ae ed on myco hizal Fm plan s pe o med wo se han hose ed on Nm, showing highe mo ali y, lowe weigh and impai ed de elopmen (Figu es 2B,D,F). The esul s e eal ha he e ec o myco hiza ion on la al pe o mance depends on P a ailabili y, as MIR was obse ed a he mode a e (0.7 mM) P le els, bu no a he low (0.3 mM) one. Rema kably, a simila pa e n was obse ed in he in e ac ion wi h B. cine ea. Again, MIR was only obse ed a 0.7 mM P, bu no a 0.3mM P, and while he e ec o P was significan in Nm plan s (Nm 0.3 s Nm 0.7 - es , p<0.0001), i was no significan in Fm plan s (Fm 0.3 s Fm 0.7 - es , p= 0.24) (Figu e 3A). Di e ences in MIR a e no di ec ly ela ed o changes in he nu i ional s a us o he plan To add ess whe he he P-dependen e ec s o myco hiza on la al pe o mance we e due o nu i ional aspec s, we e alua ed plan biomass, an hocyanin and nu ien con en s. Shoo biomass was dependen on P e iliza ion, and g ow h p omo ion by myco hiza depended on P le els: Fm p omo ed plan g ow h a low P, while no plan g ow h p omo ion was obse ed a 0.7 mM P (Figu e 3B). An hocyanins we e only influenced by P le els (Figu e 3C). Rega ding he nu i ional alue o he lea es, P con en in lea es inc eased wi h P e iliza ion, and i was significan ly highe in myco hizal plan s (Figu e 3D). P e iliza ion also significan ly influenced ca bon (C) and ni ogen (N) le els in lea es, inc easing C and educing N concen a ion a 0.7mM as compa ed o 0.3 mM. While myco hiza ion did no ha e a global e ec on hei con en , he in e ac ion be ween P and M was significan o C con en , wi h myco hiza displaying highe C le els han Nm plan s unde he low P e iliza ion (Figu es 3E,F), while he in e ac ion be ween P and M was significan in C con en . As myco hiza ion had he same e ec on he nu ien con en o lea es a he highes P le el (highe P alues, no changes in C no N as compa ed o Nm plan s), myco hiza- ela ed changes in he nu i ional alue o lea es do no seem o explain he di e en ial impac o myco hiza ion on la al pe o mance unde he di e en P e iliza ion le els. Mos mic onu ien s we e significan ly influenced by P (Ca, C , Cu, Fe, Mg, Mn, Mo, Na, Ni, S, S and Zn), while M influenced only some o hem (as Cu, Fe, K, Li, Mn, S , Zn) (Table S3). Only K was significan ly egula ed by he in e ac ion o he wo ac o s (Table S3). P a ailabili y impac s he le els o de ense- ela ed phy oho mones and gene exp ession We explo ed whe he he impac o P le els on MIR was ela ed o di e en ial ac i a ion o plan de ense esponses. In o de o moni o ea ly de ense esponses, and aiming o educe he a iabili y associa ed wi h pa hogen de elopmen o di e en ial eeding by he la ae, we used OGs as elici o s. Plan s g own in pa allel o hose in he whole plan he bi o y Dejana e al. 10.3389/ pls.2022.1060926 F on ie s in Plan Science on ie sin.o g07 assay ( hus same age, g owing condi ions and myco hizal coloniza ion le els) we e challenged by sp aying a ully expanded lea wi h an OG solu ion, as desc ibed in Gami e al. (2020). T ea ed lea es we e analyzed o ho mone con en and de ense- ela ed gene exp ession 6 hou s a e OG applica ion. The le els o jasmonic acid (JA), i s p ecu so OPDA, auxin (IAA) and abscisic acid (ABA), as he majo ho mones in ol ed in plan esponses o chewing he bi o es and nec o ophic pa hogens, and desc ibed o be egula ed by OGs, we e e alua ed (Figu e 4). Mul iway ANOVA confi med a significan impac o P e iliza ion on he OPDA, ABA and IAA le els, bu no on he JA con en . AM symbiosis only impac ed B CD EF A FIGURE 2 Impac o myco hiza on he bi o e pe o mance unde di e en P a ailabili y. S. exigua pe o mance o la ae ed on lea es o myco hizal (Fm, do ed line) and non-myco hizal (Nm, con inuous line) plan s e ilized wi h 0.3 o 0.7 mM o P, ligh and da k g ey, espec i ely; (n=9). Six- weeks pos -inocula ion wi h F. mosseae (Fm), plan s we e in es ed wi h second ins a S. exigua la ae ( wo pe plan , n=18 pe ea men ) using a clip-cage o confine he la ae o a leafle . In es a ion was main ained o h ee weeks by mo ing he clip-cage e e y wo days. (A, B) S. exigua mo ali y (C, D) weigh and (E, F) indi iduals eaching pupa s age. S a is ical analyses we e pe o med independen ly o each P e iliza ion le el: (A, E) 0.3 mM and (B, F) 0.7 mM. (A–F) P alues in he uppe igh co ne o each g aph indica e s a is ically di e ences in mo ali y and pupa ion be ween Nm and Fm, acco ding o Log- ank (Man el-Cox) es . Fo la al biomass (C, D), alues a e he weigh mean o su i ed la ae ± SD. As e isks indica e significan di e ences be ween Nm and Fm ea men s a gi en ime poin acco ding o - es . ns: no significan ; +:p < 0.1; *:p < 0.05; **:p < 0.01; ***:p < 0.001; ****:p<0.0001. Dejana e al. 10.3389/ pls.2022.1060926 F on ie s in Plan Science on ie sin.o g08 he IAA le els, while OG ea men significan ly a ec ed all ho mone le els excep o ABA. No ewo hy, no significan in e ac ion be ween he di e en ac o s was obse ed o ho mones excep o ABA le els. O e all, P deficiency significan ly educed OPDA and IAA le els, bu inc eased ABA. OGs enhanced JA, OPDA and IAA le els ega dless o he myco hizal s a us o he plan (Figu e 4). Myco hiza ion only had a significan e ec on IAA le els, al hough i modula ed he impac o P and OG on ABA le els (Figu e 4). Salicylic acid (SA) le els we e also de e mined, showing a educ ion by inc easing P o OG ea men , bu no significan e ec o myco hiza o ac o s in e ac ion was obse ed (Figu e S2). As ho mone le els we e no di e en in lea es o myco hizal plan s, we hypo hesized ha myco hizal plan s could p ime downs eam de ense esponses, bu ha his e ec was dependen on P le els. We analyzed he gene exp ession o well cha ac e ized JA egula ed an i-he bi o e de ense ma ke s, including he Leucyl aminopep idase A (LapA), P o einase inhibi o II (PinII), Th eonine deaminase (TD)andMul icys a in (MC)(Uppalapa i e al, 2005); Yan e al, 2013. Su p isingly, in non-challenged plan s (-OG) all hese de ense genes (excep MC) we e up- egula ed in Nm plan s g own unde low P le els as compa ed o Nm g own unde mode a e P le els, bu his up egula ion was no obse ed in myco hizal Fm plan s (Figu es 5A–E;Tables S4 and S5). OG ea men inplan sg ownunde lowP esul edina educed exp ession o hese genes in Nm plan s, while hey showed a sligh induc ion in Fm plan s (see old changes in Table S5). In con as , unde mode a e (0.7 mM) P le els, hese genes showed simila exp ession le els in myco hizal and non-myco hizal plan s in he absence o challenge; howe e hey we e significan ly induced by OG ea men only in Fm plan s. Thus, he exp ession analyses confi med a p imed esponse o myco hizal plan s o he OG ea men unde su ficien P (Figu es 5A–D;Table S5). A simila p imed esponse was ound o he gene encoding a de ense polygalac u onase inhibi ing p o ein (LePGIP), also ela ed o de ense esponses (Ba oncelli e al., 2016)(Figu e 5E). The educed le els o de ense genes in non-challenged (-OG) myco hizal plan s unde P s a a ion, led us o explo e he exp ession o egula o s o JA-dependen de ense esponses. JAZ p o eins a e key nega i e egula o s o JA signaling, ep essing BC DEF A FIGURE 3 Impac o P e iliza ion and myco hiza ion on he nu i ional alue o oma o lea es. Bo y is cine ea in ec ion, shoo biomass, an hocyanin, and nu ien con en s in oma o lea es o non-myco hizal (Nm) and myco hizal oma o plan s colonized by (F)mosseae (Fm). Plan s we e e ilized by wo P concen a ions: 0.3 mM and 0.7 mM P. (A) Diame e o nec o ic lesions 3 days pos inocula ion wi h (B) cine ea in de ached adul lea es om oma o (n=9). (B) Shoo esh weigh (n=9), (C) an hocyanin (n=6), (D) phospho us (n=6), (E) ca bon (n=6) and (F) ni ogen (n=6) con en we e measu ed in oma o plan s 6 weeks pos myco hizal inocula ion. Da a ep esen he means o n independen biological eplica es ± SD. Two-way ANOVA wi h myco hizal (M) and P ea men s (P) as ac o s, was pe o med, and he significance o he ac o s and hei in e ac ion is indica ed in he uppe igh co ne o each g aph. Di e en le e s ep esen s a is ically significan di e ences (ANOVA, Fishe ’s Leas Significan Di e ence (LSD) es ; p<0.05) whe e he in e ac ion be ween ac o s was obse ed. O he wise, as e isks deno e significan e ec o a ac o and hei in e ac ion. ns: no significan ; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001. Dejana e al. 10.3389/ pls.2022.1060926 F on ie s in Plan Science on ie sin.o g09 SUPPLEMENTARY TABLE 5 Fold changes in gene exp ession. Colo s indica e up- egula ion ( ed) and down- egula ion (blue) and in ensi ies a e de e mined by he in ensi y o he changes: ligh colo >1.5 o <0.75 and da k colo >2 o <0.5 and significan e ec s a e highligh ed in bold. (A) E ec o P s a a ion on gene exp ession in Nm plan s ( old change in Nm and Fm plan s e ilized a 0.3 s 0.7 mM). (B) E ec o OG elici a ion on gene exp ession in Fm o Nm plan s g own a 0.3 mM and 0.7 mM P ( old change +OG/-OG). (C) E ec o AM symbiosis on gene exp ession a 0.3 mM and 0.7 mM P ( old change Fm/Nm). Bold numbe s and as e isks indica e significan di e ences ( - es , p<0.05) be ween (A) 0.3 s 0.7 mM, (B) +OG s -OG and (C) Fm s Nm. +: p<0.1; *: p<0.05; **: p<0.01; ***: p<0.001. SUPPLEMENTARY TABLE 6 Mul iway ANOVA o gene exp ession da a. E ec s o he di e en ac o s: AM symbiosis (M), P e iliza ion (P) and elici a ion (OG ea men , OG) and hei in e ac ions we e analyzed. Da a ep esen alues and significan e ec s a e highligh ed in bold. As e isks indica e significan di e ences: +: p<0.1; *: p<0.05; **: p<0.01; ***: p<0.001. Re e ences Aljbo y, Z., and Chen, M. S. (2018). Indi ec plan de ense agains insec he bi o es: A e iew. Insec Sci. 25, 2–23. doi: 10.1111/1744-7917.12436 Ande sen, C. L., Jensen, J. L., and Ø n o , T. F. (2004). 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