Analyse van de afweermechanismen van tomaat tegen infecties van bladsnijwonden door Botrytis cinerea

Botrytis cinerea infects a large number of horticultural crops, including fruits, vegetables and ornamentals. One of the economically most import hosts of B. cinerea is tomato. All aboveground tomato plant parts can be infected by this pathogen. However, different plant organs display varying degrees of susceptibility. In tomato crops grown in open-air or unheated greenhouses B. cinerea infections of fruits and leaves are responsible for major losses. As such, tomato fruit and leaf infections by B. cinerea have been the subject of extensive research in the fields of phytopathology and plant physiology. On the other hand, in the intensive tomato cropping systems in high-tech, heated greenhouses, which are typical for Western and Northern Europe, Canada and the Northern USA, stem infections by B. cinerea are the main problem. Such stem infections originate from wounds that are created by the necessary cultural practice of pruning of the oldest leaves. After initial penetration of the leaf pruning wounds, growth of the pathogen results in killing of the host cells and eventually an expanding stem lesion. Once the lesion reaches the vascular tissues in the stem, transport of water and nutrients from the roots to the productive part of the plant is inhibited. This quickly causes the infected plant to wilt, with complete losses of the remaining yield as a consequence. However, preliminary results indicated that not all types of leaf pruning wounds are equally susceptible to infection by B. cinerea. The current study focusses on the difference in susceptibility between smooth stem wounds (SSW), where the leaf is completely removed flush to the stem, and pruning wounds on which a small petiole stub was left behind (PS). First, we characterized the difference in susceptibility between SSW and PS to spontaneous infections by naturally present B. cinerea inoculum in a commercial tomato greenhouse. We observed that SSW display absolute resistance to spontaneous infections, while PS are susceptible. The same difference in susceptibility was also observed when leaf pruning wounds were artificially inoculated. Nevertheless, B. cinerea was able to infect SSW at a low incidence in the latter experimental setup when conditions for infection were favourable. Next, we showed that the susceptibility of both SSW and PS decreases if inoculation is delayed for several hours after wounding. When inoculation was delayed for 48 hours after wounding, B. cinerea could no longer successfully infect either wound type. Since conidia of this pathogen require the moisture on the wound surface to germinate, it could be hypothesized that the increased resistance is the result of a decreasing availability of water on the wound surface over time. By using infrared imaging and image analysis we were able to demonstrate that the latter decrease in susceptibility over time is not associated with wound drying. Evaporation of water from the wound surface was continuously and consistently observed until well beyond 48 hours post wounding, at which time B. cinerea was already no longer able to cause infection.Tomato defence against infections of fruits and leaves by B. cinerea are reportedly largely based on inducible defence mechanisms, which are under the control of disease signalling pathways. We thus tested the phenotypes for susceptibility of SSW and PS to infection by B. cinerea of signalling mutants and transgenic lines that had been previously demonstrated to be affected in resistance against tomato leaf or fruit infections. While preventing salicylic acid accumulation by overexpressing the NahG gene had no significant effect, the susceptibility of PS was increased in the ethylene insensitive mutant never ripe and decreased in the jasmonic acid and abscisic acid biosynthesis mutants defenceless1 and sitiens, respectively. However, no altered phenotype for resistance of SSW was observed in any of the tested mutants or transgenic lines. As such, it could be concluded that the increased resistance of SSW as compared to PS does not depend on these 3 major disease signalling pathways.To broaden the search for the molecular basis behind the increased resistance of SSW, a transcriptome analysis was performed. RNA (cDNA) isolated from samples of eight different wound type-treatment combinations was hybridized to tomato microarrays, being the unwounded, non-inoculated tissues that are situated at the location where (i) SSW and (ii) PS would be created; wounded and mock inoculated (iii) SSW and (iv) PS; wounded and B. cinerea inoculated (v) SSW and (vi) PS; and the abscission zones of wounded and (vii) mock or (viii) B. cinerea inoculated PS. A large impact of wounding and mock inoculation on the transcriptomes of both SSW and PS was observed. Several putative defence-associated biological processes were induced in both wound types, including response to biotic stress, cell wall modifications and hormone metabolism. However, only minor differences were observed between SSW and PS. Furthermore, the putative defence-related transcriptome changes appeared to be broader and more intense in PS than in SSW. The additional effect of B. cinerea inoculation on the changes already induced by wounding (and mock inoculation) was small, regardless of the wound type. As such, it was considered unlikely that the transcriptional reprogramming induced by wounding or pathogen recognition are responsible for the difference in susceptibility between SSW and PS. However, when comparing the transcriptomes of SSW and PS before wounding or pathogen inoculation, higher basal expression levels were discovered in SSW than in PS for a wide range of putative defence-related genes. It was thus hypothesized that the increased resistance of SSW is caused by the high constitutive expression of defence-related genes.Eight putative defence-related genes were selected based on their higher basal expression levels in SSW than in PS, according to the transcriptome data. The selected genes were an aspartic protease gene (ASP), two genes coding for eight-cysteine motif proteins (TPRP-F1 and DIR3), three genes encoding PR5 family proteins (TLP, OLP1 and OLP2), the gene for PROTEINASE INHIBITOR II (PINII) and a chitinase encoding gene (CHI17). For each gene an expression profile in SSW, PS, stem epidermis, stem cross sections, leaves, flowers and shoot apexes was determined by qRT-PCR. Basal expression levels for every gene were at least 2-fold higher in SSW than in PS and differences determined by qRT-PCR corresponded to those observed in the microarray approach. Furthermore, overall basal expression of the selected genes was low in leaves and high in flowers and shoot apexes. These findings are in agreement with the predicted expression profile of genes related to plant defence.Finally, virus induced gene silencing (VIGS) was used to individually silence the expression of the selected genes. Plants were infiltrated with Agrobacterium tumefaciens strains carrying the respective DNA constructs and transverse stem sections were excised. The residual expression levels of the target genes in the stem pieces was determined by qRT-PCR and only plant sets with an average residual expression of the target gene of less than 50% were considered for susceptibility phenotyping. Inoculation of transverse stem sections of plants with reduced transcript levels of TPRP-F1 or CH17 with B. cinerea resulted in significantly increased lesion size as compared to control stem pieces. This indicates a role for the latter genes in tomato defence against B. cinerea and supports the hypothesis that genes with high basal expression levels in SSW might indeed contribute to the increased resistance of the latter wound type.

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