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The type III effector AvrXv3 from Xanthomonas campestris pv. vesicatoria (Xcv) elicits a resistance response in the tomato line Hawaii 7981. To test whether similar genes participate in responses triggered by recognition of different avirulence proteins, we examined the effect of AvrXv3 expression on the plant transcriptome as compared to that of other avirulence proteins. By microarray analysis we monitored expression of approximately 8,600 tomato genes upon inoculation with isogenic Xcv strains differing only by the avrXv3 gene. Changes in transcript levels of 139 genes were observed within 8 h, and a massive shift in expression of 1,294 genes was detected at 12 h. Recognition of AvrXv3 modulated a large number of genes encoding transcription factors and signaling components. In addition, genes involved in defense and stress responses, lipid metabolism, protein degradation, and secondary metabolism were mainly up-regulated. Conversely, genes related to photosynthesis and protein synthesis were generally down-regulated. Many novel genes encoding proteins of unknown function were also identified. A comparison between AvrXv3-modulated genes and those differentially expressed in tomato plants recognizing other bacterial effectors revealed partial overlap and similar distribution in functional classes. The identification of tomato genes modulated by AvrXv3 expression paves the way for dissecting defense networks activated by recognition of this effector in resistant plants.

Transcriptome profiling via cDNA microarray analysis identified 869 genes that are differentially expressed in developing tomato (Solanum lycopersicum) pericarp. Parallel phenotypic and targeted metabolite comparisons were employed to inform the expression analysis. Transcript accumulation in tomato fruit was observed to be extensively coordinated and often completely dependent on ethylene. Mutation of an ethylene receptor (Never-ripe [Nr]), which reduces ethylene sensitivity and inhibits ripening, alters the expression of 37% of these 869 genes. Nr also influences fruit morphology, seed number, ascorbate accumulation, carotenoid biosynthesis, ethylene evolution, and the expression of many genes during fruit maturation, indicating that ethylene governs multiple aspects of development both prior to and during fruit ripening in tomato. Of the 869 genes identified, 628 share homology (E-value ≤1 × 10−10) with known gene products or known protein domains. Of these 628 loci, 72 share homology with previously described signal transduction or transcription factors, suggesting complex regulatory control. These results demonstrate multiple points of ethylene regulatory control during tomato fruit development and provide new insights into the molecular basis of ethylene-mediated ripening.

International audience; Gene expression profiling holds tremendous promise for dissecting the regulatory mechanisms and transcriptional networks that underlie biological processes. Here we provide details of approaches used by others and ourselves for gene expression profiling in plants with emphasis on cDNA microarrays and discussion of both experimental design and downstream analysis. We focus on methods and techniques emphasizing fabrication of cDNA microarrays, fluorescent labeling, cDNA hybridization, experimental design, and data processing. We include specific examples that demonstrate how this technology can be used to further our understanding of plant physiology and development (specifically fruit development and ripening) and for comparative genomics by comparing transcriptome activity in tomato and pepper fruit.

A novel method for detection and identification of specific alleles has been developed utilizing immobilized mismatch binding protein (IMBP). The assay involves the use of biotin-labeled probes, which are prepared by PCR amplification of cloned fragments with known sequence. The use of probes avoids many of the problems associated with the extreme sensitivity of IMBP assays to errors in PCR amplification. The method can be used to monitor PCR fidelity and to genotype both diploid and haploid organisms and has been used to distinguish rifampicin-sensitive and -resistant strains of Mycobacterium tuberculosis and to detect and distinguish two alleles of the sheep prion protein gene involved in susceptibility to scrapie.

An accurate and highly sensitive mutation detection assay has been developed. The assay is based on the detection of mispaired and unpaired bases by immobilized mismatch binding protein (Escherichia coli MutS). The assay can detect most mismatches and all single base substitution mutations, as well as small addition or deletion mutations. The assay is simple to use and does not require the use of either radioactivity or gel electrophoresis.

The Xanthomonas campestris pv. vesicatoria avirulence gene avrRxv specifies resistance on the tomato line Hawaii 7998 by interacting with three nondominant plant resistance genes. AvrRxv molecular properties that impinge on its avirulence activity were characterized and transcriptional changes caused by AvrRxv expression in resistant tomato plants were extensively examined. AvrRxv localized predominantly to the cytoplasm and possibly in association with plasma and nuclear membranes in both resistant and susceptible tomato plants. The AvrRxv cysteine protease catalytic core was found to be essential for host recognition, because introduction of mutations in this domain affected the ability of AvrRxv to elicit a hypersensitive response and the inhibition of bacterial growth in resistant plants. In addition, expression profiles were analyzed for approximately 8,600 tomato genes in resistant plants challenged with X. campestris pv. vesicatoria strains expressing wild-type AvrRxv or a catalytic core AvrRxv mutant. In all, 420 genes were identified as differentially modulated by the expression of a functional AvrRxv, including over 15 functional classes of proteins and a large number of transcription factors and signaling components. Findings of this study allow the development of new hypotheses about the molecular basis of recognition between AvrRxv and the corresponding resistance proteins, and set the stage for the dissection of signaling and cellular responses triggered in tomato plants by this avirulence factor.