Host Specificity and Virulence Mechanisms of Xanthomonas Type III Effector Proteins in Bacterial Spot Disease

Xanthomonas spp are the causative agents of bacterial spot disease on cultivated pepper, Capsicum annuum, and tomato, Solanum lycopersicum. Although pepper and tomato are closely related in the Solanaceae, four species of xanthomonads have differing host specificities and utilize unique virulence strategies between these two crops plants. A major factor differentiating these pathogens are the Type III Effector (T3E) proteins they deploy to overcome the plant’s immune system and increase the host’s susceptibility. The genetic diversity and composition of T3E repertoires in a large sampling of field strains have yet to be explored on a genomic scale, limiting our understanding of pathogen evolution in an agricultural setting. To this end, we sequenced the genomes of sixty-seven Xanthomonas euvesicatoria (Xe), Xanthomonas perforans (Xp), and Xanthomonas gardneri (Xg) strains isolated from diseased pepper and tomato fields in the southeastern and midwestern United States. T3E repertoires were computationally predicted for each strain and whole genomic phylogenies were employed to understand better the genetic relationship of strains in the collection. From this analysis we detected a division in the Xp population that supported a model whereby a host-range expansion of Xp field strains on pepper is due, in part, to a loss of the T3E AvrBsT. Xp-host compatibility was further studied with the observation that a double deletion of the T3Es AvrBsT and XopQ allowed a gain of host range for Nicotiana benthamiana. Additionally, a single deletion of XopQ expanded the host range of Xe to N. benthamiana, while Xg was a natural pathogen of N. benthamiana. Extensive sampling of field strains and an improved understanding of effector content will aid in efforts to design plant disease resistance strategies targeted against highly conserved effectors./nXg has emerged recently as the dominant tomato pathogen in parts of the United States and South America. It is responsible for severe crop losses and causes spotting on fruits. Furthermore, Xg appears to be spreading globally. In its repertoire of Type III effectors, Xg possesses a single Transcription Activator Like (TAL) effector protein, AvrHah1, which has previously been shown to confer enhanced water soaked lesions in pepper. TAL effectors act as transcription factors that manipulate expression of target host genes to increase host susceptibility. We investigated the molecular mechanism of AvrHah1-dependent water soaking and the effects of water soaking on enhancing disease severity in tomato. We observed that water from outside the leaf was drawn into the apoplast in Xg-, but not XgΔAvrHah1-, infected tomato, and that water soaking can serve as a mechanism to “ferry” new bacteria into the apoplast. Additionally, AvrHah1 increased the bacterial population present on the surface of diseased tomato leaves. Comparing the transcriptomes of tomato infected with Xg wt vs XgΔAvrHah1 revealed that thousands of genes were differentially upregulated in the presence of AvrHah1. We identified two highly upregulated basic Helix Loop Helix (bHLH) transcription factors with predicted Effector Binding Elements (EBEs) as direct targets of AvrHah1. We mined our RNA-seq data for genes that were highly upregulated but without EBEs and identified two pectin modification genes, a pectate lyase and pectinesterase, which are expressed in response to the bHLH transcription factors and are therefore indirect targets of AvrHah1. Importantly, designer TAL effectors (dTALEs) for the bHLH transcription factors and the pectate lyase complement water soaking in XgΔAvrHah1. By modifying the plant cell wall to enhance water uptake and increase tissue damage, AvrHah1 may improve bacterial dispersal from the apoplast and thereby enhance disease transmission. Understanding lesion development may improve the design of disease tolerance in crops by reducing symptom development and overall pathogen transmission.

Advisor: Staskawicz, Brian J