Host resistance mechanisms and fungal infection strategies in the Botrytis cinerea-tomato interaction

The broad-host fungal pathogen B. cinerea can infect more than 1000 plants and is causing severe economic losses worldwide. However, the current knowledge of the plant-B. cinerea interaction, as outlined in Chapter I, is insufficient to adequately control the grey mould disease and therefore more research efforts are needed.So far there are no tomato genotypes reported that display full resistance to B. cinerea. Wild tomato relatives such as S. habrochaites LYC4 possess partial resistance to B. cinerea and are important genetic resources that can be exploited to improve the B. cinerea resistance in cultivated tomato, which can only be implemented once we obtain a better understanding of the resistance mechanisms (Chapter VII). B. cinerea inoculation under lab conditions requires supplementation with nutrients in the inoculum to enable efficient and synchronized infection (Benito et al., 1998). When the inoculum was constituted of 1000 spores/µl suspension in PDB medium, most of the inoculation droplets on both MM and LYC4 leaves formed expanding lesions (compatible interaction). In this situation, the partial resistance in LYC4 was manifested as smaller lesions as compared to MM. We also tested a different type of minimal medium consisting of salts, 10 mM sucrose and 10 mM potassium phosphate. Under this inoculation condition, most of the inoculation droplets in LYC4 formed black dispersed non-expanding lesions (considered to be resulting from an “incompatible interaction”), whereas, the majority of droplets could still form expanding lesions in MM. Remarkably, increasing the sucrose concentration to 50 mM restored the compatible interaction in LYC4. We hypothesized that the nutrient-rich condition was favorable for fungal infection and as a result could mostly overcome the plant defenses in the early stage of infection and lead to a compatible interaction. The use of low sugar concentration medium on the contrary reduced the aggressiveness of B. cinerea. This rendered the B. cinerea infection on the one hand frequently contained in partially resistant LYC4 and on the other hand sufficiently aggressive to break through the resistance in MM and to cause spreading lesions. The controlled, predictable high incidence of incompatible interaction in LYC4 (non-expanding lesions) using the 10 mM sucrose inoculation medium enabled a relatively “black and white” condition to better investigate the resistance mechanisms from LYC4 even if it is conferred by multiple QTLs with minor effects.In Chapter IV, we performed RNA-seq analysis to investigate the global transcriptional changes in both plant and fungus during B. cinerea infection in LYC4 and MM. More importantly, the use of different sucrose concentrations in the inoculum (10 mM vs 50 mM) in LYC4 leaves which led to incompatible and compatible interactions, respectively, were also analyzed by RNA-seq. In agreement with previously published reports of large scale transcriptional reprogramming in plant-B. cinerea interactions, we observed that around 20% of the genes in the LYC4 genome were differentially expressed at 24 hpi. Besides, we observed that LYC4 and MM differ in the expression of a large number of genes in the absence of B. cinerea. For instance, before B. cinerea inoculation, there were > 8000 differentially expressed genes (approximately equal numbers of up-regulated and down-regulated genes) in the leaf samples of LYC4 compared with MM. Thus, the deciphering of the resistance mechanism is challenging but is still under investigation. Further perspectives to facilitate the study of B. cinerea resistance in LYC4 were proposed in Chapter VI.Remarkably, delayed induction of plant cell death by B. cinerea coupled with delayed and attenuated plant responses were revealed during LYC4 infection (Chapter III). Toxic compounds, possibly accumulated in the trichomes of LYC4 leaves, are potentially playing a role in the interaction of wild tomato-B. cinerea interactions as indicated by the strong induction of fungal detoxification genes in the early infection stages on LYC4 (Chapter IV).The other part of this thesis focused on the specific role of an important antimicrobial compound, α-tomatine, in the interaction of tomato and B. cinerea.  The background information prior to this thesis project is reviewed in Chapter IV. So far three types of tomatinase have been reported in tomato pathogens that catalyze the hydrolysis of different glycoside bonds. As described in Chapter VII, tolerance mechanisms to plant antimicrobial compounds in fungal pathogens are often under transcriptional regulation. Therefore, we performed a RNA-seq analysis to reveal B. cinerea genes involved in tolerance to α-tomatine. We identified several α-tomatine-responsive genes including the BcTom1 encoding GH43 tomatinase and a GT28a gene encoding a glycosyl transferase. We demonstrated their contribution in tolerance to α-tomatine and unveiled their role in tomato infection (Chapter V). This study not only filled the current knowledge gap about genes encoding tomatinase activities but also identified novel non-hydrolytic mechanisms for tolerance to α-tomatine. Besides, we believe that the RNA-seq strategy to identify transcriptional response in plant pathogens against diverse plant antimicrobial compounds can broaden our horizon of plant-microbe interactions in the future.To study the role of α-tomatine in basal defense in tomato plants, we deleted two α-tomatine biosynthetic genes GAME4 (Solyc12g006460) and GAME2 (Solyc07g043410) separately by CRISRP/Cas9 genome editing (Chapter VI). The deletion of GAME4 abolished the α-tomatine biosynthesis. However, GAME2 knockout mutants still accumulated α-tomatine at similar concentration as the wild-type plants. This result indicates that this GAME2 gene, which was proclaimed to catalyze the β1-tomatine UDP-xylosyltransferase, is not essential for α-tomatine biosynthesis in tomato, despite the fact that it resides in a biosynthetic gene cluster with other GAME genes. Therefore future studies will focus on the identification of the real glycosyltransferase responsible for the last glycosylation step of α-tomatine synthesis. Besides, GAME4-KO plants displayed increased susceptibility against B. cinerea and Phytophthora infestans in initial infection assay indicates that α-tomatine contributes to disease resistance in tomato. However, the role of α-tomatine in tomato basal defense needs to be addressed with more repetitions and with more plant pathogens as discussed in Chapter VI.&nbsp