ENGLISH ABSTRACT: In the field of agriculture, plant pathogens are a major concern because of the severe damage these organisms cause to crops yearly. Fundamental studies regarding plant pathogens and their modes of action made it possible for researchers in the field of molecular biology to investigate pathogens further on a molecular level. Botrytis cinerea, has been used to great effect as a model system to investigate various aspects regarding pathogenesis, also on a molecular level.
Molecular research done on B. cinerea over the last few years has shown that the endopolygalacturonases (EPGs) of this fungus are key role players in pathogenesis. This hydrolytic enzyme family of six members, encoded by the Bcpg1-6 genes, are important in breaking down the complex cell wall polymers of host plants, enabling the fungus to penetrate its host sufficiently. It has been shown that both BcPG1 and 2 are crucial for virulence of B. cinerea. A leucine-rich repeat inhibitor protein situated in the cell wall of various plant species, the polygalacturonase-inhibiting protein (PGIP), has been proven to interact with and inhibit EPGs, and thus the necrotic actions of B. cinerea. From literature it was clear that specific data regarding individual interactions of fungal EPGs with PGIPs are lacking currently. Furthermore, most experiments regarding the effects of EPG as well as interaction and inhibition studies of EPGs and PGIPs, rely on in vitro methods, without the possibility to contextualize the results on an in vivo or in planta level. The scope of this study was to specifically address the issues of individual EPG:PGIP interactions and the use of possible in vivo methodology by using EPGs from a highly virulent South African strain of B. cinerea and the grapevine VvPGIP1 that has been previously isolated in our laboratory. This PGIP, originally isolated from Vitis vinifera cv Pinotage, has been shown to inhibit a crude EPG extract from this strain with great efficiency. The approach taken relied on heterologous over-expression of the individual Bcpg genes and the isolation of pure and active enzymes to evaluate the inhibition of the EPGs with VvPGIP1. The genes were all successfully over-expressed in Saccharomyces cerevisiae with a strong and inducible promoter, but active enzyme preparations have been obtained only for the encoding Bcpg2 gene, as measured with an agarose diffusion assay. The in vitro PGIP inhibition assay is also based on the agarose diffusion assay and relies on activity of the EPGs to visualize the inhibiting effect of the PGIP being tested. The active EPG2, however, was not inhibited by VvPGIP1 when tested with this assay. The EPG encoding genes from B. cinerea were transiently over-expressed also in Nicotiana benthamiana by using the Agrobacterium-infiltration technique. Transgene expression was confirmed by Northern blot analysis and EPG-related symptoms were observed five to eight days post-infiltration. Differential symptoms appeared with the various EPGs, providing some evidence that the symptoms were not random events due to the infiltration or a hypersensitive response. Moreover, the symptoms observed for EPG2 was similar to those that were reported recently by another group on the same host. In spite of the expression data and the clear symptoms that developed, active preparations, as measured with the agarose diffusion plate asay, could only be obtained for EPG2 again.
In our search for a possible in vivo method to detect and quantify EPG activity and inhibition by PGIPs, we tested and evaluated a technique based on chlorophyll fluorescence to detect the effect of EPGs on the rate of photosynthesis. Our results showed that the over-expression of these genes reduced the rate of electrons flowing through photosystem II, indicating metabolic stress occurring in the plant. We used the same technique to evaluate possible interaction between VvPGIP1 respectively with BcPG1 and 2 and found that the co-expressing of the Vvpgip1 gene caused protection of the infiltrated tissue, indicating inhibition of EPG1 and 2 by VvPGIP1. For EPG2, the observed interaction and possible inhibition by VvPGIP1 is the first report to our knowledge of an interaction between this specific EPG2 and a PGIP. Moreover, to further elucidate the in planta interaction between VvPGIP1 and the EPGs from the South African B. cinerea strain, we tested for possible interactions by making use of a plant two-hybrid fusion assay, but the results are inconclusive at this stage.
Previous studies in our laboratory have shown that several natural mutations exist between PGIP encoding genes from different V. vinifera cultivars. Based on this finding and the fact that these natural mutations could result in changes with regard to EPG inhibition and ultimately disease susceptibility, we isolated an additional 37 PGIP encoding genes from various grapevine genotypes, some of which are known for their resistance to pathogens.
Combined, these results make a valuable contribution to understand plant pathogen interactions, specifically in this case by modeling the interactions of pathogen and plant derived proteins. The possibility to use in vivo methods such as chlorophyll fluorescence to follow these interactions on an in planta level, provides exciting possibilities to strenghten and contextualize in vitro results.
AFRIKAANSE OPSOMMING: Plantpatogene organismes veroorsaak jaarliks erge skade aan landbougewasse en word dus as ’n ernstige probleem in die landbousektor beskou. Diepgaande studies wat handel oor plantpatogene en hul metodes van infeksie het dit vir molekulêre bioloë moontlik gemaak om patogene nou ook op molekulêre vlak verder te bestudeer. Botrytis cinerea is baie effektief as modelsisteem gebruik om verskeie aspekte van patogenese verder te bestudeer, ook op ‘n molekulêre vlak.
Molekulêre navorsing op B. cinerea, het getoon dat die endopoligalakturonases (EPGs) van dié swam kernrolbelangrik in patogenese is. Hierdie sesledige hidrolitiese ensiemfamilie word gekodeer deur die Bcpg1-6 gene en is belangrik vir die afbraak van die komplekse selwandpolimere van plantgashere, om suksesvolle gasheerpenetrasie te veroorsaak. Daar is aangetoon dat beide BcPG1 en 2 essensieël vir virulensie van die patogeen is. ’n Leusienryke-herhalings inhibitorproteïen wat in die selwand van verskeie plantspesies voorkom, die poligalakturonase-inhiberende proteïen (PGIP), het interaksie met en inhibeer EPGs en gevolglik ook die nekrotiserende aksies van B. cinerea. Uit die literatuur is dit duidelik dat spesifieke inligting aangaande individuele interaksies van fungiese EPGs met PGIPs tans nog ontbreek. Verder word daar op in vitro metodologie staatgemaak wannneer die effekte van EPGs asook die interaksie en inhibisie met PGIPs bestudeer word, sonder om die konteks van die in vivo- of in planta-omgewing in ag te neem. Die fokus van hierdie studie was om aspekte van individuele EPG:PGIP interaksies, asook die moontlike gebruik van in vivo metodologie te bestudeer deur EPGs, afkomstig van ’n hoogs virulente Suid-Afrikaanse ras van B. cinerea en die wingerd VvPGIP1, wat vroeër in ons laboratorium geïsoleer is, te gebrruik. Hierdie PGIP wat uit Vitis vinifera cv Pinotage geïsoleer is, inhibeer ’n kru EPG-ekstrak van bogenoemde ras baie effektief. Die benadering wat gevolg is het op die ooruitdrukking van die individuele Bcpg-gene in heteroloë sisteme staatgemaak en die gevolglike isolering van suiwer en aktiewe ensieme om EPG-inhibisie deur VvPGIP1 te beoordeel. Al die gene is suksesvol in Saccharomyces cerevisiae ooruitgedruk onder ’n sterk induseerbare promotor, maar volgens ’n agarose-diffundeerbare toets kon aktiewe ensiempreparate slegs vir die enkoderende Bcpg2 verkry word. Die in vitro PGIP-inhibisie toets is ook op die gemelde toets gebasseer en vereis EPG-aktiwiteit om die inhiberende effek van die PGIP, te visualiseer. Die aktiewe EPG2 is egter nie deur VvPGIP1 geïnhibeer met die aanleg van hierdie toets nie. Die EPG-enkoderende gene van B. cinerea is ook tydelik in Nicotiana benthamiana ooruitgedruk deur gebruik te maak van ’n Agrobacterium-infiltrasietegniek. Transgeenuitdrukking kon met die Noordelike kladtegniek bevestig word en EPG-verwante simptome is vyf tot agt dae na infiltrasie waargeneem. Verskillende simptome vir die verskillende EPGs is waargeneem, wat aanduidend is dat die simptome nie lukrake gevolge van die infiltrasies, of ’n hipersensitiewe respons is nie. Verder kon die simptome wat EPG2 vertoon het, gekorreleer word met dié wat onlangs deur ’n ander groep op dieselfde gasheer waargeneem is. Ten spyte van die ekspressiedata en die waargenome simptome, kon aktiewe ensiempreparate op die agarose-diffundeerbare toets, weereens slegs vir EPG2 waargeneem word.
’n Metode wat gebasseer is op chlorofilfluoressensie is getoets en geëvalueer as ’n moontlike in vivo metode om EPG aktiwiteit en inhibisie deur PGIPs waar te neem en te kwantifiseer. Die resultate het bevestig dat die ooruitdrukking van hierdie gene die elektronvloeitempo deur fotosisteem II verminder het wat ’n aanduiding is dat metaboliese stres in die plant heers. Dieselfde tegniek is gebruik om die moontlike interaksies tussen BcPG1 en 2 en VvPGIP1 te bestudeer en het aangetoon dat die mede-uitdrukking van die Vvpgip1-geen aanleiding gee tot ’n beskermende effek van die geinfiltreerde weefsel, wat aanduidend is van inhibisie van EPG1 en 2 deur VvPGIP1. In die geval van EPG2 is hierdie interaksie en moontlike inhibisie met ’n PGIP die eerste waarneming in die verband. In ’n verdere poging om die in planta-interaksie tussen VvPGIP1 en die EPGs van die Suid-Afrikaanse B. cinerea ras uit te klaar, is ’n plantgebasseerde twee-hibriede toets aangelê, maar geen klinkklare resultate kon verkry word nie.
Vorige werk het bevestig dat verskeie natuurlike mutasies in PGIP-enkoderende gene, afkomstig van verskillende V. vinifera kultivars, voorkom. Hierdie resultaat en die feit dat hierdie mutasies verskille in EPG inhibisie en uiteindelik vatbaarheid vir siektes kan beïnvloed, het aanleiding gegee tot die isolering van ’n verdere 37 PGIP-enkoderende gene uit ‘n verskeidenheid druifplantgenotipes, sommige waarvan juis bekend vir hul weerstand teen patogene is.
Die gekombineerde resultate wat in dié studie verkry is, maak ’n waardevolle bydrae tot die verstaan van plant-patogeeninteraksies, spesifiek met die modelering van interaksies van patogeen- en plantgebasseerde proteïene. Die moontlikheid om in vivo-metodes soos chlorofilfluoressensie te gebruik in in planta-analises, is besonder bemoedigend om in vitro-resultate te versterk en ook in konteks te plaas.
91 leaves : ill.
Phytopathogenic microorganisms -- Molecular aspects