The Cytological And Transcriptomic Investigations Into Interactions Of Tomato And Powdery Mildew

Tomato powdery mildew (Oidium neolycopersici) is a worldwide fungal disease of tomato(Solarium lycopersicum). So far, five dominant, one recessive resistance gene and three major resistanceQTLs (R-QTLs) have been discovered and mapped or fine-mapped on the tomato genome.We have investigated the fungus-induced transcriptome by differential gene expression profilingusing cDNA-AFLP of mock- and inoculated susceptible, monogenic and polygenic resistant genotypes(Chapter 2). Our results showed that in the tomato-O. neolycopersici interaction, twice as many genesare induced in the compatible interaction as in the incompatible interactions. Genes involved in basaldefense of the compatible interaction and R-gene/QTL mediated resistance response in the incompatibleinteractions largely overlap. About 40ï¼…of these "common" genes display earlier expression in theincompatible interactions compared to the compatible interaction, while the remaining genes show asimilar temporal pattern of expression in both interactions. Transcripts of the sequenced differentiallyexpressed TDFs (DE-TDFs) that are earlier up-regulated in the incompatible interactions compared tothe compatible interaction or that are resistance specific, predominantly execute putative roles in plantdefense and signal transduction. By contrast, transcripts of the sequenced DE-TDFs showing similartemporal patterns in compatible and incompatible interactions are often associated with housekeepingfunctions and regulation (Chapter 2). Therefore, we propose that the host plants employ similarcomponents of the defense pathways during both compatible and incompatible interactions of tomatoand O. neolycopersici, and that the timing difference in the expression of these components contributesto the final outcome of the interactions (Chapter 2).A set of near or nearly isogenic lines (NILs) carrying the dominant R genes Ol-1, Ol-3, Ol-4, Ol-5and Ol-6, the recessive gene ol-2, and different combinations of R-QTLs were previously developed.These lines have been used for microscopic analysis of the infection process (Chapter 4) and for thestudy of the fungus-induced transcriptome (Chapter 3 and Chapter 5). Microscopic observations revealedthat the recessive gene ol-2, dominant Ol genes and different combinations of R-QTLs confer resistanceto O. neolycopersici at different infection stages of the interaction.Microscopic investigations of infected leaves of NILs carrying different R genes/QTLsdemonstrated that HR is involved in R-QTL mediated resistance responses and NILs with differentindividual R-QTL(s) display different types of necrotic cells upon the fungal inoculation. Penetratedpapillae, vesicle accumulation and changes in the extra-haustorial matrix are also associated withR-QTL-mediated resistance responses, but are not specific to individual R-QTL(s). The pyramiding ofR-QTL(s) into a single tomato line results in a high-level resistance comparable to Ol-1-mediatedmonogenic dominant resistance, mainly because of a high incidence of a novel type of necrotic cells(Chapter 4). Our results suggest as well that the genetic background can influence the R-QTL mediatedresistance, since we found that different tomato lines with the same combination of R-QTLs displaydifferent resistance phenotypes. For instance the macroscopic lesion size and incidence on inoculatedleaflets are different in N3Qa and N3Qb, which both have all three R-QTLs (Chapter 4). A smaller lesion is associated with a faster reaction, thus less costly for the plant. Therefore, multi-R-QTL tomato plantswith suitable genetic backgrounds could be selected in breeding programs through evaluation of themacroscopic lesion size after inoculation.Tomato lines containing the recessive R gene ol-2, the dominant R genes Ol-1 and Ol-4 mediateresistance responses involving papilla formation, slow HR and fast HR respectively. The resistanceresponses to powdery mildew (O. neolycopersici) controlled by these genes were investigated usingcDNA-AFLP and reverse transcription PCR (RT-PCR). The results indicate that the transcript profiles ofthese responses are different. Generally, the transcript profiles of Ol-1 mediated resistant and susceptibleresponses are similar. A large part of the common up-regulated genes in both the Ol-1 mediatedresistance and the compatible interaction (S-MM) was not induced in the Ol-4 mediated resistanceresponse and not or later up regulated in the ol-2 mediated resistance response. Sequence information ofa small number of differential expressed transcript derived fragments (DE-TDF) specific to Ol-4 andol-2 mediated resistance responses further suggests that these responses are different from the Ol-1mediated resistance response. The RT-PCR analyses indicated that the ol-2 mediated resistance involvingpapilla formation is independent of SA, JA and ethylene pathways. Therefore, we suggest that the OI-2protein is a compatibility factor necessary for the successful establishment of compatibility betweentomato and O. neolycopersici, or that ol-2-mediated resistance employs another defense pathway. TheOl-4 mediated resistance response is associated with the ethylene pathway but not JA and SA. Anisoform of lipoxygenase (LOX), likely other than 13 LOX, plays a role in both Ol-4 (fast HR) and ol-2(papilla formation) mediated resistance responses. Ol-1 mediated resistance appears to be associatedwith both SA and ethylene pathways (Chapter 3).We also analyzed the pathogen-induced transcript profiles of tomato lines containing differentcombinations of R-QTLs to O. neolycopersici, and compared those with the profiles of the S-MM and aNIL carrying the dominant R gene Ol-1, using cDNA-AFLP. About 4,000 bands were displayed with 78selected primer combinations. In total, 204 DE-TDFs were induced upon O. neolycopersici inoculationand 72 DE-TDFs displayed a differential expression level between NILs that was not dependent oninoculation. Transcripts that show similar timing in both compatible and incompatible interactions wereassociated with basal defense or establishment of compatibility, probably the result of the response ofsuccessfully attacked cells. Transcripts that display earlier/higher expression in the incompatibleinteractions compared to the compatible interaction showed a systemic induction as well. Salicylic acid(SA) and H₂O₂ might be important diffusive signals for both monogenic and polygenic resistance inthese tomato NILs. The small fraction of differentially expressed genes specific to (partially) resistantgenotypes may fine-tune the activation of defense pathways in resistant genotypes through regulatingtranscription and translation. Pyramiding of R-QTLs into a single tomato line leads to a high-levelresistance comparable to that mediated by Ol-1, and generally the same defense pathways are triggeredby these combined R-QTLs compared to individual R-QTLs. We propose that pyramiding of R-QTLsonly alters the defense pathways quantitatively rather than qualitatively. The map locations of elevensequenced DE-TDFs were in-silico determined. One of them could be a good candidate for Ol-1 or R-QTLI because of its specific expression to and co-localization with Ol-1 and R-QTL1.The combined results of Chapters 2-5 are discussed in relation to relevant literature. Wedemonstrated that tomato defense barriers to O. neolycopersici are correlated with different infectionstages during the interaction. Conservation and quantitative nature of pathogen-induced transcriptomesof compatible and slow HR (Ol-1/R-QTL) mediated incompatible interactions of tomato and O.neolycopersici are proposed. Additionally, transcriptome changes during the resistance mechanismsmediated by fast HR (Ol-4) and by papilla formation (ol-2) suggest different resistance mechanisms thatare proposed. Genes required for the resistance mediated by R genes/QTLs in tomato are hypothesized.