Erial fruit blotch, elucidating the mechanisms of fruit infection by Acidovorax citrulli

Abstract

Bacterial fruit blotch (BFB) of cucurbits is caused by the Gram-negative bacterium Acidovorax citrulli. BFB affects cucurbit production worldwide, and mainly watermelon and melon. Most A. citrulli strains are divided into two genetically differentiated groups: while group I strains have been mainly associated with melon and other non-watermelon cucurbits, group II strains are more aggressive on watermelon. Like many Gram-negative plant-pathogenic bacteria, A. citrulli relies on a functional type III secretion system (T3SS) for pathogenicity. The T3SS is responsible for direct secretion of bacterial protein effectors to the host cell. Type III-secreted effectors (T3Es) contribute to virulence through manipulation of the host cell metabolism and suppression of plant defense. Our previous collaboration showed that group I and II strains significantly differ in their T3E arsenal (Eckshtain-Levi et al., Phytopathology 2014, 104:1152-1162). Using comparative genomics, we also showed that group I and II strains of A. citrulli have substantial differences in their genome content (Eckshtain-Levi et al., Front. Microbiol. 2016, 7:430). Our long-term goals are to identify the genetic determinants that contribute to virulence and host preferential association of the two major groups of A. citrulli, and to exploit these insights to develop effective BFB management strategies. We hypothesize that differences in the arsenal of T3Es, are greatly responsible for the differences in host preferential association between strains belonging to the two groups. The specific objectives of this project were: (1) to investigate the susceptibility of cucurbit species to group I and II strains under field conditions; (2) to assess the contribution of T3Es and other virulence factors to A. citrulli virulence and host preference; and (3) to characterize the mechanisms of action of selected T3Es of A. citrulli. In the frame of objective 1, we carried out three field experiments involving inoculation of several cucurbit crops (watermelon, melon, pumpkin and squash) with group I and II strains. Findings from these experiments confirmed that A. citrulli strains exhibit a preference for watermelon and melon. Moreover, we demonstrated, for the first time under field conditions, host-preferential association of group I and II strains to melon and watermelon, respectively. While host-preferential association was observed in leaves and in fruit tissues, it was more pronounced in the latter. In this part of the project we also developed a duplex PCR assay to differentiate between group I and II strains. In the frame of objective 2, we employed a multifaceted approach combining bioinformatics and experimental methods to elucidate the T3E arsenal of A. citrulli. These experiments led to discovery that A. citrulli strains possess large arsenals with more than 60 T3E genes. Remarkably, we found that ~15% of the T3E genes are group-specific. Advances were achieved on the contribution of selected T3E genes and other virulence determinants to the ability of A. citrulli to colonize the fruit and other tissues of melon and watermelon. Last, in the frame of objective 3, we advanced our understanding on the mode of action of few key T3Es of A. citrulli. We also optimized a virus-induced gene silencing (VIGS) system for functional genomics in melon and watermelon. This system will allow us to test melon and watermelon genes that may have defense or susceptibility roles related to BFB disease. Overall, this collaboration substantially enriched our knowledge on basic aspects of BFB disease. We believe that the fruits of this collaboration will greatly contribute to our ultimate goal, which is generation of durable resistance of melon and watermelon to A. citrulli.