A functional genomics approach to dissect resistance of tomato to bacterial spot disease

Abstract

The research problem. Bacterial spot disease in tomato is of great economic importance worldwide and it is particularly severe in warm and moist areas affecting yield and quality of tomato fruits. Causal agent of spot disease is the Gram-negative bacterium Xanthomonas campestris pv. vesicatoria (Xcv), which can be a contaminant on tomato seeds, or survive in plant debris and in association with certain weeds. Despite the economic significance of spot disease, plant protection against Xcvby cultural practices and chemical control have so far proven unsuccessful. In addition, breeding for resistance to bacterial spot in tomato has been undermined by the genetic complexity of the available sources of resistance and by the multiple races of the pathogen. Genetic resistance to specific Xcvraces have been identified in tomato lines that develop a hypersensitive response and additional defense responses upon bacterial challenge. Central goals of this research were: 1. To identify plant genes involved in signaling and defense responses that result in the onset of resistance. 2. To characterize molecular properties and mode of action of bacterial proteins, which function as avirulence or virulence factors during the interaction between Xcvand resistant or susceptible tomato plants, respectively. Our main achievements during this research program are in three major areas: 1. Identification of differentially expressed genes during the resistance response of tomato to Xcvrace T3. A combination of suppression subtractive hybridization and microarray analysis identified a large set of tomato genes that are induced or repressed during the response of resistant plants to avirulent XcvT3 bacteria. These genes were grouped in clusters based on coordinate expression kinetics, and classified into over 20 functional classes. Among them we identified genes that are directly modulated by expression of the type III effector protein AvrXv3 and genes that are induced also during the tomato resistance response to Pseudomonas syringae pv. tomato. 2. Characterization of molecular and biochemical properties of the tomato LeMPK3MAP kinase. A detailed molecular and biochemical analysis was performed for LeMPK3 MAP kinase, which was among the genes induced by XcvT3 in resistant tomato plants. LeMPK3 was induced at the mRNA level by different pathogens, elicitors, and wounding, but not by defense-related plant hormones. Moreover, an induction of LeMPK3 kinase activity was observed in resistant tomato plants upon Xcvinfection. LeMPK3 was biochemically defined as a dual-specificity MAP kinase, and extensively characterized in vitro in terms of kinase activity, sites and mechanism of autophosphorylation, divalent cation preference, Kₘand Vₘₐₓ values for ATP. 3. Characteriztion of molecular properties of the Xcveffector protein AvrRxv. The avirulence gene avrRxvis involved in the genetic interaction that determines tomato resistance to Xcvrace T1. We found that AvrRxv functions inside the plant cell, localizes to the cytoplasm, and is sufficient to confer avirulence to virulent Xcvstrains. In addition, we showed that the AvrRxv cysteine protease catalytic core is essential for host recognition. Finally, insights into cellular processes activated by AvrRxv expression in resistant plants were obtained by microarray analysis of 8,600 tomato genes. Scientific and agricultural significance: The findings of these activities depict a comprehensive and detailed picture of cellular processes taking place during the onset of tomato resistance to Xcv. In this research, a large pool of genes, which may be involved in the control and execution of plant defense responses, was identified and the stage is set for the dissection of signaling pathways specifically triggered by Xcv.