Transcriptomic analysis of disease resistance responses using a tobacco-Botrytis cinerea pathosystem

Basson, Carin Elizabeth (2017-12)

Thesis (PhDAgric)--Stellenbosch University, 2017.

Thesis

ENGLISH SUMMARY: Cultivation of plants for food and raw materials is regularly hampered by phytopathogens that reduce quality and yield. Necrotrophic fungi are among the most damaging pathogens, killing plant cells to complete their lifecycle. Many of these fungi release cell wall degrading enzymes (CWDE) to breach this first defensive barrier of plant cells. Some of the most important CWDE are polygalacturonases (PGs), that degrade the pectin-component of the plant cell wall and often act as virulence factors. To combat PGs, plants have evolved polygalacturonase-inhibiting proteins (PGIPs). In most cases, constitutive expression of PGIPs confer some level of resistance to pathogenic fungi. Two mechanisms have been proposed to explain PGIP-induced resistance. Firstly, they protect the cell wall from degradation through PGIP-PG inhibition interactions and secondly, they assist in activating defence signalling, by prolonging the survival of signalling molecules (oligogalacturonides, OGs) derived from cell wall degradation to activate the salicylic acid (SA) branch of defence signalling. The defence role of Vitis vinifera PGIP1 (VviPGIP1) was established using transgenic tobacco (Nicotiana tabacum) plants challenged with Botrytis cinerea, a model necrotrophic fungal phytopathogen. VviPGIP1 inhibited the two PGs that are virulence factors for B. cinerea, and reduced lesion diameter during infection assays. A subsequent characterisation of uninfected transgenic tobacco revealed wide-ranging changes in gene expression, enhanced lignin deposition and changes in hemicellulose composition, pointing to new and previously unexplored alterative functions of PGIPs in plant defence. Moreover, when the tobacco lines were infected, a more rapid accumulation of the defence hormone jasmonic acid was seen in the transgenic lines. These changes suggested that the constitutive expression of VviPGIP1 induced defence priming, a mechanism whereby plants induce slight metabolic and transcriptomic changes prior to infection, but display an enhanced defence response upon challenge. The work that formed part of this study follows on from these previous studies and had as aim to study the mechanisms that contributed to the defence phenotype observed in the tobacco lines expressing the grapevine PGIP encoding gene. The approach was to profile the molecular response of the host (tobacco) when infected by B. cinerea, contrasting the native tobacco response with that of the PGIP tobacco lines. The disease progression of B. cinerea has been extensively studied in Arabidopsis and other model host species. The first two days after infection has been established as the critical phase that determines the outcome of the interaction (susceptibility/resistance). Consequently, this period was chosen to profile transcriptional changes following B. cinerea infection of wild-type and VviPGIP1-expressing tobacco. A time-course, consisting of five sampling points (0 h, 12 h, 24 h, 36 h and 48 h), was used to profile the localised defence response and the response in distal organs. The time-course represented two day-night cycles, providing the opportunity to investigate diurnal gene expression patterns. Wild-type tobacco mounted a localised defence response that shared many elements with those of other plant hosts of B. cinerea, including the dampening of diurnal patterns and induction of antioxidant mechanisms, jasmonate and ethylene biosynthesis and secondary metabolism. In leaves distal to the infection, the diurnal patterns of gene expression were not disrupted, but genes related to anti-fungal proteins and secondary metabolite synthesis were induced. This suggested partial induced resistance (IR) had been activated by distal infection, but systemic acquired resistance (SAR) had not yet been established. Profiling of volatile secondary metabolites emphasised a strong ontogenic effect. The resistant tobacco line (expressing VviPGIP1) displayed enhanced activation of jasmonate/divinyl ether biosynthesis and repression of ethylene-responsive transcription factors. Monolignol biosynthesis was affected, and may have led to altered lignin composition. Several biological processes were affected at 24 hours after infection, reportedly a critical point during B. cinerea pathogenesis. These included enhanced activation of pterostilbene synthesis, fungitoxic SAR8.2 proteins, proteinase inhibitors and antimicrobial peptides, while oxidative stress was reduced. In terms of priming, several stress-responsive genes were more rapidly induced in the PGIP line, which also displayed an accelerated transition from source to sink metabolism. With regards to the specific role of VviPGIP1 during infection, this study represented the first untargeted transcriptomic analysis of an infected PGIP-expressing tobacco line. The enhancement of jasmonate synthesis suggested that hormone signalling may differ between VviPGIP1-expressing transgenics and plants expressing cotton or bean PGIPs. In leaves distal to the infection, where signalling molecules derived from cell wall degradation by B. cinerea would not be generated, priming- and IR-like responses were observed, further underscoring the connection of VviPGIP1 with defence priming. The recurring appearance of cell wall modification in responses to B. cinerea, and the prior analyses that found changes in hemicellulose composition, prompted a more detailed examination of the xyloglucan endotransglycosylase/hydrolase (XTH) family in tobacco. The catalytic domain and other characteristic features of XTHs were identified for predicted XTH proteins. Functional information from characterised XTHs was mapped onto homologous sequences in order to infer functions for tobacco XTHs, however, the high sequence homology of XTHs in general, and of XTHs with contrasting expression patterns, suggested that promoter analysis would be required to accurately predict functions for specific XTHs. The sequence alignments and transcriptional information generated for the XTH gene family during this study provides a useful context for studies into tobacco cell wall metabolism. This study has generated novel gene expression data for B. cinerea-infected tobacco, provided the opportunity to compare the timing and magnitude of transcriptional responses in susceptible and resistant plant lines, and to investigate the basis for PGIP-induced resistance. Further studies should consider utilising de novo sequencing to identify processes not represented on the microarray and attempt to distinguish between OG-induced responses and PGIP-induced responses. This study successfully reinforced the proposed defence priming role of VviPGIP1, not only at the site of infection, but in tissues where PGs were not active.

AFRIKAANS OPSOMMING: Gehalte- en opbrengste van oesplante word dikwels belemmer deur plant patogene. Nekrotrofiese swamme is van die mees skadelike patogene; hierdie organismes maak plantselle dood terwyl hul lewensiklus voltooi word. Baie van hierdie swamme stel selwand-afbrekende ensieme (SWAE) vry om die selwand, die plant se eerste verdegingslinie, te oorkom. Poligalakturonases (PGs) is van die mees prominente SWAE en is verantwoordelik om die pektien-komponent van die plant selwand af te breek, maar word ook dikwels as virulensiefaktore beskou. Om PGs te beheer het plante poligalakturonase-inhiberende proteïene (PGIPs) ontwikkel. In die meeste gevalle lei konstitutiewe uitdrukking van PGIPs tot 'n mate van weerstand teen patogeniese swamme. Twee meganismes is voorgestel om dié weerstand te verduidelik. Eerstens beskerm hulle die selwand deur middel van PGIP-PG inhibisie interaksies, en tweedens help hulle om verdedigingseine te aktiveer, deurdat hulle aksies die leeftyd verleng van seinmolekules wat hul oorsprong het as afbreekprodukte van selwande. Dié seinmolekules, oligogalakturonides (OGs), aktiveer die salisielsuur (SA)-been van die verdedigingsein-netwerk. Die verdedigingsrol van Vitis vinifera PGIP1 (VviPGIP1) is bevestig met behulp van transgeniese tabak (Nicotiana tabacum) plante wat met Botrytis cinerea, 'n model-nekrotrofiese plant swampatogeen, infekteer is. VviPGIP1 het die twee PGs wat virulensiefaktore vir B. cinerea is geïnhibeer, en die deursnee van infeksieletsels tydens infeksie verminder. In 'n opvolgstudie is bevind dat ongeïnfekteerde transgeniese tabak grootskaalse veranderinge in geen-uitdrukking, verhoogde lignienvorming en veranderinge in hemisellulose-samestelling vertoon het. Dit het gedui op unieke funksies van PGIPs in plantverdediging. Verder, tydens infeksie het die transgeniese tabaklyne vinniger toenames in jasmoonsuur getoon. Hierdie veranderinge het daarop gedui dat die konstitutiewe uitdrukking van VviPGIP1 verdedigingsvoorvoerding (“priming”) veroorsaak het, 'n meganisme waardeur plante slegs klein metaboliese en transkripsionele veranderinge voor infeksie ervaar, maar 'n verbeterde verdedigingsreaksie na infeksie vertoon. Die huidige studie volg dus op die werk van hierdie vorige studies en het ten doel gehad om die meganismes wat bygedra het tot die verdedigingsfenotipe van die VviPGIP1-tabaklyne verder te bestudeer. Die benadering was om die molekulêre reaksies van die gasheer (tabak) te volg tydens ‘n B. cinerea infeksie en om die kontrole tabak se reaksie met dié van die PGIP-tabaklyne te vergelyk. Die siekteverloop van B. cinerea is reeds omvattend bestudeer in Arabidopsis en ander model gasheer spesies. Die eerste twee dae na infeksie word beskou as die kritieke fase wat die uitkoms van die interaksie sal bepaal (vatbaarheid/weerstand). Gevolglik was hierdie tydperk gekies om transkripsionele veranderinge te volg tydens B. cinerea-infeksie van kontrole en ‘n VviPGIP1-uitdrukende tabaklyn in die huidige studie. 'n Tydsverloopstudie, bestaande uit vyf tydpunte (0 h, 12 h, 24 h, 36 h en 48 h), is gebruik om die reaksie op infeksie in weefsel by/naby die letsel (lokale reaksie), sowel as in blare distaal tot die infeksie (distale reaksie) te volg. Die tydsverloop het twee dag-nag siklusse ingesluit, wat die geleentheid gebied het om dag-nag (“diurnal”) uitdrukkingspatrone te ondersoek. Die resultate het getoon dat die kontrole tabak 'n lokale verdedigingsreaksie geloods het wat baie elemente gedeel het met dié van ander plantgashere van B. cinerea, byvoorbeeld die demping van dag-nag-patrone en die induksie van antioksidantmeganismes, jasmoon- en etileenbiosintese, asook sekondêre metabolisme. In blare distaal aan die infeksie is die dag-nag-patrone van geen-uitdrukking nie ontwrig nie, maar gene wat kodeer vir anti-swam proteïene en sekondêre-metaboliet sintese is geïnduseer. Dit dui daarop dat gedeeltelike geïnduseerde weerstand (IW) geaktiveer word deur distale infeksie, maar sistemiese weerstand (“systemic acquired resistance”, SAR) nog nie bereik is nie. Profiele van vlugtige sekondêre metaboliete het ook 'n sterk ontogeniese effek uitgewys. Die weerstandbiedende tabaklyn (wat VviPGIP1 uitdruk) het verhoogde aktivering van jasmonaat/divinieletersintese en onderdrukking van etileen-beheerde transkripsiefaktore getoon. Monolignolsintese is ook geaffekteer, en kon gelei het tot veranderde ligniensamestelling. Verskeie biologiese prosesse was geraak teen 24 uur na die infeksie, wat ‘n kritieke punt tydens B. cinerea-patogenese is. Dit sluit byvoorbeeld ‘n sterker aktivering van pterostilbeensintese, fungitoksiese SAR8.2 proteïene, protease-inhibeerders en antimikrobiese peptiede in, terwyl oksidatiewe stres skynbaar verminder is. In terme van verdedigings-voorbereiding, het die PGIP-lyn verskeie gene wat verband hou met stres-reaksies, vinniger geaktiveer en ook vinniger van bron- na sink-metabolisme oorgeskakel. Hierdie studie beskryf die eerste transkriptomiese analise van 'n geïnfekteerde PGIP-uitdrukkende tabaklyn. Die verbetering van jasmonatesintese wat waargeneem is kan daarop dui dat hormoonregulering van die verdedigingsein-netwerk verskil tussen plante wat VviPGIP1 uitdruk en dié wat katoen- of boontjie-PGIPs uitdruk. In blare distaal aan die infeksie, waar seinmolekules wat afgelei is van selwandafbreking deur B. cinerea nie gegenereer sou word nie, is verdedigingsvoorvoerding- en IW-agtige reaksies waargeneem, wat die koppeling van VviPGIP1 met verdedigings-voorbereiding verder onderstreep. Die herhaalde voorkoms van selwandmodifikasie in reaksies op B. cinerea, en die vorige ontledings wat veranderinge in hemisellulose-samestelling aangetref het, het 'n meer gedetailleerde ondersoek van die xiloglukaan-endotransglikosilase/hidrolase (XTH)-familie in tabak gemotiveer. Die katalitiese domein en ander kenmerkende eienskappe van XTHs is geïdentifiseer vir hipotetiese XTH proteïene. Funksionele inligting afkomstig van gekarakteriseerde XTHs is op homoloë proteïenvolgordes toegepas om sodoende moontlike funksies vir tabak XTHs af te lei. Die hoë volgorde-homologie van XTHs in die algemeen en van XTHs met kontrasterende uitdrukkingspatrone het egter daarop gedui dat die analise van geenpromotore sou nodig wees om funksies akkuraat te voorspel vir spesifieke XTHs. Die volgorde-belynings en transkripsionele inligting wat vir die XTH-geenfamilie tydens hierdie studie geskep is, bied egter 'n nuttige konteks vir studies in tabak selwandmetabolisme. Hierdie studie het nuwe gene-ekspressie data vir B. cinerea-geïnfekteerde tabak verkry; die geleentheid gebied om die tydsberekening en omvang van transkripsionele response in vatbare en weerstandige plantlyne te vergelyk; asook om die basis vir PGIP-geïnduseerde weerstand te ondersoek. Verdere studies moet oorweeg om gebruik te maak van de novo-volgordebepaling om prosesse wat nie op die mikrorooster (“microarray”) voorgestel word nie, te identifiseer en om sodoende te probeer onderskei tussen OG-geïnduseerde en PGIP-geïnduseerde reaksies. Resultate van hierdie studie het die verdedigingsrol van VviPGIP1 verder versterk en toegelig en het aangedui dat dit nie net op die plek van infeksie ‘n rol speel nie, maar ook in weefsels waar PGs nie aktief was nie.

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