| Bacterial fruit blotch (BFB), caused by Acidovorax citrulli, has been a serious seed-bornbacterial disease of water melon, melon and other cucurbits. Since1980’s, BFB has spread allover the world and caused tremendous losses to the production of cucurbits, especially thefruit production and seed industry of water melon and melon. Due to the existence ofcopper-resistant strains of A. citrulli, very limited antibacterial chemicals, and short ofresistant cultivars, BFB has been a serious threat to the cucurbit industry worldwide. However,little is known on the biology and molecular basis of BFB pathogenesis.To facilitate understanding of molecular basis of A. citrulli pathogenicity, we analyzedinfection of two subgroup strains of A. citrulli on different aboveground organs of cucumberand established a compatible A. citrulli-cucumber pathosystem. We further generated atransposon insertion mutant library of A. citrulli by diparental conjugation. Library screeningon the established A. citrulli-cucumber pathosystem led to the identification of pathogenicityaltered mutants. Finally, the function of several identified candidate genes were determinedby integrated approaches, including bioinformatics analysis and functional complementationassays. The main results of the study are as follows:1. Under experimental conditions, A. citrulli was shown to be able to infect and colonizeall aboveground organs, including fruits. Group I strains were more aggressive on cucumberthan that of group II strains. Wound inoculation with106cells suspension in5mmol·L-1PBS(pH7.4) on young true leaves (20-day-old) of cucumber was shown suitable for pathogenicitydetermination of A. citrulli on detached leaves, but repeatability was limited. Woundinoculation with107cells of group I strain in H2O on cotyledons of the6-day-old cucumberseedlings was developed to be a simple, reliable assay for evaluation of BFB severities andlarge scale screening for pathogenicity altered mutants of A. citrulli.2. Transposition in A. citrulli is difficult. To obtain insertional mutants with transposonTn5, an optimized mutagenesis protocol was developed, by extending the conjugationduration of donor and recipient cells and the conjugation in the first round of antibiotic selection, by collecting one colony from each matings followed by third round of antibioticsselection. A mutant library containing a total of2100Tn5mutants were generated for A.citrulli. Library analysis showed that the transposition insertions were single, independent,and stable. Several pathogenicity altered mutants were identified through screening themutants on cucumber, further indicating that the established cucumber-A. cirtulli pathosystemwas efficient. The generated mutants provide a valuable resource for research on biology andpathology of A. citrulli.3. Selected pathogenicity altered A. citrulli mutants were analyzed in detail. Sequencealignment and gene functional complementation assays confirmed that the predicted type IIIsecretion system ATPase in A. citrulli was a non-flagellar secretion system ATPase (HrcN).The hrcN mutant lost the ability to cause BFB symptom on cucumber and failed to induce HRin tobacco, indicating that HrcN was essential for pathogenicity and functionality of NF-T3SSin A. citrulli. Comparative sequence analysis of HrcN protein and its homologs in otherrepresentative bacterial plant pathogens revealed that the NF-T3SS of A. citrulli is close tothat of Ralstonia solanacearum and Xanthomonas campestris, but distant from that ofPseudomonas syringae and Erwinia amylovora.4. Comparative sequence analysis revealed that the predicted glucose inhibited divisionprotein A (GidA) of A. citrulli had a high similarity with its homologs in other representativebacterial plant pathogens. Sequence analysis revealed that the stop codon of GidA overlappedthe start codon of the downstream GidB gene, and genes flanking GidA were also different inA. citrulli compared with that of other bacterial pathogens, suggesting that the GidA in A.citrulli likely functions in a different way compared with its homologs in other bacterialpathogens. The GidA mutant was highly attenuated in host infection, and lost the twitchingmotility. Genetic complementation assays confirmed that GidA is a virulence factor of A.citrulli, and responsible for regulating the twitching motility. |
