Acidovorax citrulli causes bacterial fruit blotch, a disease that poses a global threat to watermelon and melon production. Despite its economic importance, relatively little is known about the molecular mechanisms of pathogenicity and virulence of A. citrulli. Like other plant-pathogenic bacteria, A. citrulli relies on a type III secretion system (T3SS) for pathogenicity. On the basis of sequence and operon arrangement analyses, A. citrulli was found to have a class II hrp gene cluster similar to those of Xanthomonas and Ralstonia spp. In the class II hrp cluster, hrpG and hrpX play key roles in the regulation of T3SS effectors. However, little is known about the regulation of the T3SS in A. citrulli. This study aimed to investigate the roles of hrpG and hrpX in A. citrulli pathogenicity. We found that hrpG or hrpX deletion mutants of the A. citrulli group II strain Aac5 had reduced pathogenicity on watermelon seedlings, failed to induce a hypersensitive response in tobacco, and elicited higher levels of reactive oxygen species in Nicotiana benthamiana than the wild-type strain. Additionally, we demonstrated that HrpG activates HrpX in A. citrulli. Moreover, transcription and translation of the type 3-secreted effector (T3E) gene Aac5_2166 were suppressed in hrpG and hrpX mutants. Notably, hrpG and hrpX appeared to modulate biofilm formation. These results suggest that hrpG and hrpX are essential for pathogenicity, regulation of T3Es, and biofilm formation in A. citrulli.

A type VI secretion system (T6SS) gene cluster has been reported in Acidovorax citrulli. Research on the activation conditions, functions, and the interactions between key elements in A. citrulli T6SS is lacking. Hcp (Hemolysin co-regulated protein) is both a structural protein and a secretion protein of T6SS, which makes it a special element. The aims of this study were to determine the role of Hcp and its activated conditions to reveal the functions of T6SS. In virulence and colonization assays of hcp deletion mutant strain Δhcp, tssm (type VI secretion system membrane subunit) deletion mutant strain Δtssm and double mutant ΔhcpΔtssm, population growth was affected but not virulence after injection of cotyledons and seed-to-seedling transmission on watermelon. The population growth of Δhcp and Δtssm were lower than A. citrulli wild type strain Aac5 of A. citrulli group II at early stage but higher at a later stage. Deletion of hcp also affected growth ability in different culture media, and the decline stage of Δhcp was delayed in KB medium. Biofilm formation ability of Δhcp, Δtssm and ΔhcpΔtssm was lower than Aac5 with competition by prey bacteria but higher in KB and M9-Fe³⁺ medium. Deletion of hcp reduced the competition and survival ability of Aac5. Based on the results of Western blotting and qRT-PCR analyses, Hcp is activated by cell density, competition, ferric irons, and the host plant. The expression levels of genes related to bacterial secretion systems, protein export, and several other pathways, were significantly changed in the Δhcp mutant compared to Aac5 when T6SS was activated at high cell density. Based on transcriptome data, we found that a few candidate effectors need further identification. The phenotypes, activated conditions and transcriptome data all supported the conclusion that although there is only one T6SS gene cluster present in the A. citrulli group II strain Aac5, it related to multiple biological processes, including colonization, growth ability, competition and biofilm formation.

Acidovorax citrulli (Ac) is the causal agent of bacterial fruit blotch (BFB), and BFB poses a threat to global watermelon production. Despite its economic importance, the molecular mechanisms underlying Ac pathogenicity and virulence are not well understood, particularly with regard to its type III secreted effectors. We identify a new effector, AopP, in Ac and confirm its secretion and translocation. AopP suppresses reactive oxygen species burst and salicylic acid (SA) content and significantly contributes to virulence. Interestingly, AopP interacts with a watermelon transcription factor, ClWRKY6, in vivo and in vitro. ClWRKY6 shows typical nuclear localization, and AopP and ClWRKY6 co-localize in the nucleus. Ac infection, SA, and the pathogen-associated molecular pattern flg22^Ac promote ClWRKY6 production, suggesting that ClWRKY6 is involved in plant immunity and SA signaling. Furthermore, ClWRKY6 positively regulates PTI and SA production when expressed in Nicotiana benthamiana. Importantly, AopP reduces ClWRKY6 mRNA and ClWRKY6 protein levels, suggesting that AopP suppresses plant immunity by targeting ClWRKY6. In summary, we identify a novel effector associated with the virulence mechanism of Ac, which interacts with the transcription factor of the natural host, watermelon. The findings of this study provide insights into the mechanisms of watermelon immune responses and may facilitate molecular breeding for bacterial fruit blotch resistance.

LuxR‐type regulators regulate many bacterial processes and play important roles in bacterial motility and virulence. Acidovorax citrulli is a seedborne bacterial pathogen responsible for bacterial fruit blotch, which causes great losses in melon and watermelon worldwide. We identified a LuxR‐type, nonquorum sensing‐related regulator, AcrR, in the group II strain Aac‐5 of A. citrulli . We found that the acrR mutant lost twitching and swimming motilities, and flagellar formation. It also showed reduced virulence, but increased biofilm formation and growth ability. Transcriptomic analysis revealed that 394 genes were differentially expressed in the acrR mutant of A. citrulli , including 33 genes involved in flagellar assembly. Our results suggest that AcrR may act as a global regulator affecting multiple important biological functions of A. citrulli.

Bacterial fruit blotch (BFB), caused by Acidovorax citrulli, seriously affects watermelon and other cucurbit crops, resulting in significant economic losses. However, the pathogenicity mechanism of A. citrulli is not well understood. Plant pathogenic bacteria often suppress the plant immune response by secreting effector proteins. Thus, identifying A. citrulli effector proteins and determining their functions may improve our understanding of the underlying pathogenetic mechanisms. In this study, a novel effector, AopN, which is localized on the cell membrane of Nicotiana benthamiana, was identified. The functional analysis revealed that AopN significantly inhibited the flg22-induced reactive oxygen species burst. AopN induced a programmed cell death (PCD) response. Unlike its homologous protein, the ability of AopN to induce PCD was dependent on two motifs of unknown functions (including DUP4129 and Cpta_toxin), but was not dependent on LXXLL domain. More importantly, the virulence of the aopN mutant of A. citrulli in N. benthamiana significantly decreased, indicating that it was a core effector. Further analysis revealed that AopN interacted with watermelon ClHIPP and ClLTP, which responds to A. citrulli strain Aac5 infection at the transcription level. Collectively, these findings indicate that AopN suppresses plant immunity and activates the effector-triggered immunity pathway.

LuxR-type transcriptional regulators are essential for many physiological processes in bacteria, including pathogenesis. Acidovorax citrulli is a seedborne bacterial pathogen responsible for bacterial fruit blotch, which causes great losses in melon and watermelon worldwide. However, the LuxR-type transcriptional factors in A. citrulli have not been well studied, except for the previously reported LuxR-type regulatory protein, AcrR, involved in regulating virulence and motility. Here, we characterized a second LuxR-type regulator, AclR, in the group II strain Aac-5 of A. citrulli by mutagenesis, virulence and motility assays, and transcriptomic analysis. Deletion of aclR resulted in impaired twitching and swimming motility and flagellar formation and diminished virulence but increased biofilm formation. Transcriptomic analysis revealed that 1,379 genes were differentially expressed in the aclR mutant strain, including 29 genes involved in flagellar assembly and 3 involved in pili formation, suggesting a regulatory role for AclR in multiple important biological functions of A. citrulli. Together, our results not only indicate that AclR plays a global role in transcriptional regulation in A. citrulli influencing motility, biofilm formation, and virulence but also provide perspective regarding the regulatory network of biological functions in A. citrulli. [ ] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .