Chemotropic Assay for Testing Fungal Response to Strigolactones and Strigolactone-Like Compounds

Depósi o de In es igación de la Uni e sidad de Se illa h ps://idus.us.es/ This is an Accep ed Manusc ip o an a icle published by Sp inge Na u e in S igolac ones in May 2021, a ailable a : h ps://doi.o g/10.1007/978-1-0716-1429-7_9 © 2021 Sp inge Science+Business Media, LLC, pa o Sp inge Na u e Chap e 9 1 Chemo opic assay o es ing ungal esponse o s igolac ones and 2 s igolac ones-like compounds 3 Rocío Pineda-Ma os1, An onio Di Pie o2, Da id Tu à3,* 4 1Uni e si y o Se ille, School o Ag icul u al Enginee ing, A ea o Ag o o es y 5 Enginee ing, Se ille, Spain; [email p o ec ed] 6 2Uni e si y o Có doba, Depa men o Gene ics, Có doba, Spain; [email p o ec ed] 7 3,*Uni e si y o Naples Fede ico II, Depa men o Ag icul u al Sciences, Naples, I aly; 8 da u [email protected] 9 10 Running Head: Fungal chemo opic sensing o SLs 11 12 Abs ac 13 Cu en knowledge on he mechanism o s igolac ones (SLs) as signalling 14 molecules du ing speci ic in e ac ions in he hizosphe e is mainly ela ed o he 15 con ol o ge mina ion o pa asi ic weed seeds and hyphal b anching o a buscula 16 myco hizal ungi. Thus, he ole o plan sec e ed SLs in egula ing he g ow h and 17 de elopmen o oo -colonizing ungi s ill emains con o e sial. Fusa ium oxyspo um 18 can sense and espond o ex acellula signals h ough o ien ed ge m ube 19 eme gence and edi ec ioning o hyphal g ow h owa ds g adien s o nu ien s, sex 20 phe omones o plan oo exuda es. Howe e , chemoa ac an ac i i y o SLs 21 agains mic oo ganisms li ing in he soil has no been es ed so a . He e we 22 p opose a quan i a i e chemo opic assay o unde s and i and how soil ungi could 23 sense g adien s o SLs and SLs-like sou ces. In he example case o F. oxyspo um, 24 hyphae o ungal ep esen a i e mu an s p e e en ially g ow owa ds he syn he ic SL 25 analogue GR24; and his chemo opic esponse equi es conse ed elemen s o he 26 ungal in asi e g ow h mi ogen-ac i a ed p o ein kinase (MAPK) cascade. 27 28 Key Wo ds 29 Chemo opism, ungal signalling, Fusa ium oxyspo um, GR24, oo -colonizing ungi 30 31 1. In oduc ion 32 Al hough phy oho mones and g ow h egula o s a e no mally p oduced in plan s a 33 low concen a ions o modula e sel -g ow h and physiology, he e a e se e al 34 examples in na u e o plan -in e ac ing mic obes (i.e. ungi and bac e ia) ha ei he 35 p oduce he same o e y simila chemical compounds o modula e plan g ow h o 36 esponses o hei own bene i (1-2). In ecen yea s, a g owing body o e idence has 37 shown ha phy oho mones migh unc ion as signalling messenge s, used by plan s 38 o unde g ound o abo eg ound ec ui men o bene icial o ganisms o by 39 pa hogens o loca e he plan hos (3-8). One such example a e s igolac ones (SLs), 40 a g oup o ca o enoid-de i ed compounds exhibi ing egula o y ac i i y on plan 41 g ow h and physiology, ha also igge ge mina ion o pa asi ic plan s and symbiosis 42 o a buscula myco hizal ungi (3,4,6,9) (see also Chap e s 6 and 7). Howe e , he 43 po en ial ole o plan sec e ed SLs o hei syn he ic analogues in egula ing g ow h 44 and de elopmen o o he soil-inhabi ing ungi, including plan pa hogens, has 45 emained con o e sial (10-16). 46 The Fusa ium oxyspo um species complex comp ises a la ge g oup o cosmopoli an 47 isola es ha li e in he soil as sap ophy es by h i ing on dead o decaying o ganic 48 ma e . Some ep esen a i es beha e as endophy es and as biocon ol agen s by 49 inducing plan esis ance and/o enhancing soil supp essi eness, while o he s a e 50 de imen al plan pa hogens causing ascula wil disease in mo e han one hund ed 51 di e en c op species (17). P e ious s udies e ealed ha F. oxyspo um, like o he 52 ilamen ous ungi, can sense and eo ien hyphal g ow h owa ds a a ie y o 53 chemical signals including nu ien s, sex phe omones and class III pe oxidases, a 54 majo chemoa ac an exc e ed om hos plan oo s (18,19). This sense-and-55 espond mechanism – common o bo h unicellula and mul icellula ungi – is 56 egula ed by complex signalling ci cui s which ely on he ac i i y o h ee- ie ed 57 p o ein kinase modules e med mi ogen-ac i a ed p o ein kinase (MAPK) cascades 58 (18,20). 59 In e es ingly, di ec ed g ow h o F. oxyspo um ge m ubes owa ds nu ien sou ces 60 such as suga s and amino acids is go e ned by he in asi e g ow h MAPK Fmk1, 61 while chemosensing o ungal phe omones and plan oo pe oxidases occu s ia he 62 unc ionally dis inc cell wall in eg i y (CWI) MAPK Mpk1 (18,19). P eceding s udies 63 ha e es ablished ha ungi wi h di e en ecological unc ions, including F. 64 oxyspo um, can de ec and deg ade di e en ypes o SLs (11,16,21). Mo eo e , a 65 quan i a i e assay on aga pla es (see Fig. 1) was applied o unde s and i and how 66 di e en mu an s o F. oxyspo um sense and ack SL g adien s, measu ing 67 chemo opism o ge m ubes owa ds GR24 (16). In his case, F. oxyspo um was 68 able o eo ien i s g ow h owa ds g adien s o GR24, a p ocess ha equi ed 69 conse ed elemen s o he in asi e g ow h MAPK cascade, bu no o he CWI 70 MAPK cascade (16). 71 In he p esen Chap e , he chemo opic assay – p e iously es ed in F. oxyspo um 72 MAPK mu an s – is desc ibed o i s ele ance on po en ial p ocedu es o: (i) sc een 73 ungal mu an s o wo k ou he SL pe cep ion, signalling and esponse pa hway in 74 ungi inhabi ing in he hizosphe e; and (ii) sc een SL-like molecules and analogues 75 o di e en ial chemoa ac ion ac i i y. 76 77 2. Ma e ials 78 All solu ions a e p epa ed by using s e ile dis illed ul apu e wa e (ddH2O) and 79 s o ed a oom empe a u e (~ 25°C) unless o he wise indica ed. 80 2.1 Fungal cul u es 81 1. Fungal s ains based on a collec ion o mu an s and hei wild ype isola e. 82 2. Po a o dex ose b o h (PDB) p epa ed acco ding o manu ac u e ’s 83 ins uc ions. 84 3. Flasks and magne ic s i e o p epa a ion o solu ions. 85 4. Au ocla e. 86 5. The mo egula ed o bi al shaking incuba o . 87 6. Au ocla able unnels. 88 7. Cheeseclo h (10 µm mesh). 89 8. Re ige a ed cen i uge and 50 ml cen i uge conical ubes. 90 9. S e ile ul apu e wa e . S e ilize by au ocla ing. 91 10. Mic oconidia s o age solu ion: 30% / glyce ol in wa e . Mix 30 ml o glyce ol 92 wi h 70 ml o wa e , ans e o a 250 ml sc ew-cap glass bo le and s e ilize 93 by au ocla ing. 94 2.2 Fungal chemo opism 95 1. Wa e Aga medium (WA): 0.5% (w/ ) Bac e iological Aga in ddH2O. 96 Adjus ed o pH 7 and s e ilize by au ocla ing in a sc ew-cap glass bo le. 97 S o e a oom empe a u e o 4°C. 98 2. pH me e . 99 3. Mic owa e. 100 4. Wa e ba h. 101 5. Round (90 mm diame e ) Pe i dishes. Fo chemo opic assays, p epa e he 102 Pe i dishes by d awing h ee pa allel lines wi h a 1.0 mm blue indelible 103 ma ke a he cen e o he bo om pla e, each a 0.5 cm om he o he (see 104 No e 1). Le , con ol line; cen e, sco ing line; igh , chemoa ac an line (Fig. 105 1). 106 6. Neubaue chambe o cell coun ing. 107 7. Op ical mic oscope o coun ungal conidia (400× magni ica ion) o o sco e 108 he di ec ion o ungal hyphae (200× magni ica ion). 109 8. Mic obiological lamina low hood. 110 9. SL s ock solu ion: 30 mM GR24 (syn he ic analogue o SLs) in ace one. A 30 111 µM wo king solu ion o GR24 is ob ained by dilu ing he s ock solu ion in 112 wa e . A 0.1% solu ion o ace one is used as sol en con ol. Bo h SL wo king 113 solu ion and sol en may be s o ed a −20°C un il use, bu i is ecommended 114 o p epa e he wo king solu ion esh on he day o he expe imen . 115 10. Fla -bo om plas ic boxes o chemo opic pla e incuba ion. 116 11. Fungal g ow h incuba o . 117 118 3. Me hods 119 Ca y ou all p ocedu es a oom empe a u e (~ 25°C) unless o he wise speci ied. 120 3.1 Fungal isola es, cul u e condi ions and mic oconidia collec ion 121 1. To ob ain esh ungal mic oconidia o be used in chemo opic assays, s a 122 liquid ungal cul u es by inocula ing, unde a s e ile hood, 25 µl o 123 mic oconidia suspension ( om a s ock s o ed a −80°C; see poin 4 below and 124 3.2.1) in o 250 ml E lenmeye lasks con aining 25 ml o au ocla ed PDB. 125 2. Incuba e ungal cul u es a 150 pm and 28°C o 4-5 days in an o bi al 126 shaking incuba o . 127 3. To collec mic oconidia, il e he ungal cul u e h ough ou s e ile laye s o 128 cheeseclo h in a s e ile unnel on op o a 50 ml cen i uge ube. Once he 129 conidia ha e passed h ough he il e meshes, emo e he unnel and 130 cen i uge he p ecipi a e a 6000 x g o 10 min. Ca e ully emo e he 131 supe na an and esuspend he pelle in 1 ml o s e ile ddH2O. 132 4. S o e he eshly collec ed ungal mic oconidia a −80°C in s o age solu ion o 133 use di ec ly o chemo opic assays. 134 3.2 Quan i ica ion o ungal chemo opism 135 1. Conidia concen a ion in he eshly ob ained mic oconidia solu ion is 136 es ima ed by he use o a Neubaue chambe and adjus ed o 2.5×108 137 conidia∙ml−1 wi h s e ile ddH2O (22,23). 138 2. Fungal conidia a e embedded in 4 ml WA (see No e 2) a a inal concen a ion 139 o 2.5×106 conidia∙ml−1. To do his, add 10 µl o he 2.5×108 conidia∙ml−1 140 solu ion, p epa ed in s ep 1, o e e y 4 ml o WA o ob ain he desi ed ungal 141 concen a ion (see No es 3,4); and pou i in o p e-d awn chemo opic Pe i 142 dishes (see No e 5). 143 3. Wi h a 1000-µl pipe e ip cu wo pa allel wells – espec i ely, con ol and 144 chemoa ac an well – in o he WA laye on op o he con ol and 145 chemoa ac an lines (see Fig. 1 o design). 146 4. Pipe e 40 μl o he es compound solu ion (e.g. GR24) uni o mly in o he 147 chemoa ac an well; and, 40 μl o he sol en (0.1% solu ion o ace one) in o 148 he con ol well. 149 5. Place he Pe i dishes inside a la bo om plas ic box and incuba e a 28°C in 150 he da k o 13 hou s (see No es 6-9). 151 6. Use a binocula mic oscope (200× magni ica ion) o coun he numbe o ge m 152 ube ips on he cen al sco ing line poin ing, espec i ely, owa ds he es 153 compound o he sol en con ol. Fo each chemo opic pla e, a o al o 500 154 hyphal ips a e sco ed om 5 o mo e di e en egions o he sco ing line. Fo 155 each es compound, expe imen s a e epea ed in wo o mo e independen 156 occasions. 157 7. Use he ollowing ma hema ical o mula o calcula e he chemo opic index: 158 [(H es − Hsol ) / H o al × 100] whe e, H es is he numbe o hyphae g owing owa ds he es compound; Hsol 159 is he numbe o hyphae g owing owa ds he sol en con ol and; H o al is he 160 o al numbe o hyphae coun ed. 161 162 4. No es 163 1. A oid he use o a da k-colou ed ma ke (i.e. black) o d aw he cen al 164 sco ing line on he bo om o he pla e, as opaque lines will p e en ligh 165 di usion h ough he sample and e icien isualiza ion o ungal ge m ubes 166 unde he mic oscope. As an example, S aed le Lumocolo ® pe manen pen 167 317 M, wo ks ine. 168 2. A s ock o au ocla ed WA can be s o ed a oom empe a u e o se e al 169 mon hs and used when needed. To use solidi ied WA o chemo opic assays, 170 mel he medium (in a loosened sc ew-cap bo le) in he mic owa e and keep 171 he bo le in a wa e ba h se a 45°C o a leas 1 h be o e pou ing i . 172 3. To p e en dea h o ungal p opagules, apidly mix conidia and he WA 173 solu ion in a 50 ml ube by using a 5000-µl pipe e. 174 4. A oid p epa ing mo e han 30 ml o WA plus conidia mix u e (equi alen o ~ 175 6-7 chemo opic pla es) a a ime, as his can esul in pipe e clogging due o 176 aga solidi ica ion and une en sp eading o he medium on he su ace o he 177 pla e. 178 5. Rapidly sp ead he mix u e o WA plus conidia on he pla e by epea edly 179 o a ing he pla e ho izon ally and e ically o p e en quick aga solidi ica ion 180 and une en sp eading o he medium on he su ace o he pla e. 181 6. Be ca e ul o place he Pe i dishes inside a la bo om box, making su e ha 182 hey a e pa allel o he base o he box, o p e en spilling o he 183 chemoa ac an solu ions. 184 7. W ap he plas ic box wi h aluminium oil when es ing ligh -sensi i e 185 chemoa ac an compounds 186 8. Do no mo e he box con aining he chemo opic pla es du ing he incuba ion 187 o p e en spilling o chemoa ac an solu ions. 188 9. When assaying chemoa ac an compounds ha exe a s imula o y ac i i y 189 on conidial ge mina ion o ge m ube g ow h, pla e incuba ion can be 190 pe o med a lowe ( oom) empe a u e, o o sho e ime pe iods o a oid 191 o e g ow h. 192 193 Acknowledgemen s 194