Effector CLas0185 targets methionine sulphoxide reductase B1 of Citrus sinensis to promote multiplication of 'Candidatus Liberibacter asiaticus' via enhancing enzymatic activity of ascorbate peroxidase 1.

Citrus huanglongbing (HLB) has been causing enormous damage to the global citrus industry. As the main causal agent, 'Candidatus Liberibacter asiaticus' (CLas) delivers a set of effectors to modulate host responses, while the modes of action adopted remain largely unclear. Here, we demonstrated that CLIBASIA_00185 (CLas0185) could attenuate reactive oxygen species (ROS)-mediated cell death in Nicotiana benthamiana. Transgenic expression of CLas0185 in Citrus sinensis 'Wanjincheng' enhanced plant susceptibility to CLas. We found that methionine sulphoxide reductase B1 (CsMsrB1) was targeted by the effector, and its abundance was elevated in CLas0185-transgenic citrus plants. Their interaction promoted CLas proliferation. We then determined that CsMsrB1 sustained redox state and enzymatic activity of ascorbate peroxidase 1 (CsAPX1) under oxidative stress. The latter reduced H2O2 accumulation and was associated with host susceptibility to CLas infection. Consistently, citrus plants expressing CLas0185 and CsMsrB1 conferred enhanced APX activity and decreased H2O2 content. Taken together, these findings revealed how CLas0185 benefits CLas colonization by targeting CsMsrB1, which facilitated the antioxidant activity and depressed ROS during pathogen infection.

Comparison of Copper-Tolerance Genes between Different Groups of Acidovorax citrulli.

Acidovorax citrulli populations exhibit genetic and phenotypic variations, particularly in terms of copper tolerance. Group I strains of A. citrulli generally exhibit higher copper tolerance compared to group II strains. This study aims to identify genes involved in copper tolerance to better understand the differences in copper tolerance between group I and group II strains. Representative strains pslb65 (group I) and pslbtw14 (group II) were selected for comparison. Deletion mutants of putative copper-tolerance genes and their corresponding complementary strains were constructed. The copper tolerance of each strain was evaluated using the minimum inhibitory concentration method. The results showed that the copA, copZ, cueR, and cueO genes played major roles in copper tolerance in A. citrulli, while cusC-like, cusA-like, and cusB-like genes had minor effects. The different expression levels of copper-tolerance-related genes in pslb65 and pslbtw14 under copper stress indicated that they had different mechanisms for coping with copper stress. Overall, this study provides insights into the mechanisms of copper tolerance in A. citrulli and highlights the importance of specific genes in copper tolerance.

Draft genome sequence of Xylella fastidiosa strain ATCC 35874 isolated from infected red oak in Washington, DC.

We report here the draft genome sequence of Xylella fastidiosa strain ATCC 35874. The strain was originally isolated from infected red oak in Washington, DC, and obtained from the American Type Culture Collection. The ATCC 35874 genome contains 2,543,332 bp and has a G + C content of 51.72%.

Acidovorax citrulli type III effector AopU interferes with plant immune responses and interacts with a watermelon E3 ubiquitin ligase.

Acidovorax citrulli is a seed-borne bacterium that causes bacterial fruit blotch of watermelon and other cucurbit plants worldwide. It uses a type III secretion system to inject type III effectors (T3Es) into plant cells, which affect the host immune responses and facilitate pathogen colonization. However, the current understanding of the specific molecular mechanisms and targets of these effectors in A. citrulli is limited. In this study, we characterized a novel T3E called AopU in A. citrulli group II strain Aac5, which shares homology with XopU in Xanthomonas oryzae. The Agrobacterium-mediated gene transient expression system was used to study the effect of AopU on host immunity. The results showed that AopU localized on the cell membrane and nucleus of Nicotiana benthamiana, inhibited reactive oxygen species burst induced by flg22 and the expression of marker genes associated with pathogen-associated molecular pattern-triggered immunity, but activated salicylic acid and jasmonic acid signal pathways. Further investigations revealed that AopU interacts with E3 ubiquitin ligase ClE3R in watermelon, both in vitro and in vivo. Interestingly, the deletion of aopU did not affect the virulence of A. citrulli, suggesting that AopU may have functional redundancy with other effectors in terms of its role in virulence. Collectively, these findings provide new insights into the mechanism of plant immune responses regulated by A. citrulli T3Es.

A Sec-dependent effector, CLIBASIA_04425, contributes to virulence in 'Candidatus Liberibater asiaticus'.

Citrus Huanglongbing (HLB) is the most destructive citrus disease worldwide, mainly caused by 'Candidatus Liberibacter asiaticus' (CLas). It encodes a large number of Sec-dependent effectors that contribute to HLB progression. In this study, an elicitor triggering ROS burst and cell death in Nicotiana benthamiana, CLIBASIA_04425 (CLas4425), was identified. Of particular interest, its cell death-inducing activity is associated with its subcellular localization and the cytoplasmic receptor Botrytis-induced kinase 1 (BIK1). Compared with CLas infected psyllids, CLas4425 showed higher expression level in planta. The transient expression of CLas4425 in N. benthamiana and its overexpression in Citrus sinensis enhanced plant susceptibility to Pseudomonas syringae pv. tomato DC3000 ΔhopQ1-1 and CLas, respectively. Furthermore, the salicylic acid (SA) level along with the expression of genes NPR1/EDS1/NDR1/PRs in SA signal transduction was repressed in CLas4425 transgenic citrus plants. Taken together, CLas4425 is a virulence factor that promotes CLas proliferation, likely by interfering with SA-mediated plant immunity. The results obtained facilitate our understanding of CLas pathogenesis.

pilA Gene Contributes to Virulence, Motility, Biofilm Formation, and Interspecific Competition of Bacteria in Acidovorax citrulli.

Acidovorax citrulli, the causative agent of bacterial fruit blotch, can be divided into two main groups based on factors such as pathogenicity and host species preference. PilA is an important structural and functional component of type IV pili (T4P). Previous studies have found significant differences in pilA DNA sequences between group I and group II strains of A. citrulli. In this study, we characterized pilA in the group I strain pslb65 and the group II strain Aac5. pilA mutants, complementation strains, and cross-complementation strains were generated, and their biological phenotypes were analyzed to identify functional differences between pilA in the two groups. pilA deletion mutants (pslb65-ΔpilA and Aac5-ΔpilA) showed significantly reduced pathogenicity compared with the wild-type (WT) strains; pslb65-ΔpilA also completely lost twitching motility, whereas Aac5-ΔpilA only partially lost motility. In King's B medium, there were no significant differences in biofilm formation between pslb65-ΔpilA and WT pslb65, but Aac5-ΔpilA showed significantly reduced biofilm formation compared to WT Aac5. In M9 minimal medium, both mutants showed significantly lower biofilm formation compared to the corresponding WT strains, although biofilm formation was recovered in the complementation strains. The biofilm formation capacity was somewhat recovered in the cross-complementation strains but remained significantly lower than in the WT strains. The interspecies competitive abilities of pslb65-ΔpilA and Aac5-ΔpilA were significantly lower than in the WT strains; Aac5-ΔpilA was more strongly competitive than pslb65-ΔpilA, and the complementation strains recovered competitiveness to WT levels. Furthermore, the cross-complementation strains showed stronger competitive abilities than the corresponding WT strains. The relative expression levels of genes related to T4P and the type VI secretion system were then assessed in the pilA mutants via quantitative PCR. The results showed significant differences in the relative expression levels of multiple genes in pslb65-ΔpilA and Aac5-ΔpilA compared to the corresponding WT stains. This indicated the presence of specific differences in pilA function between the two A. citrulli groups, but the regulatory mechanisms involved require further study.

Complete Genome Sequence of Erwinia amylovora Strain 99east-3-1, Isolated from Pyrus sinkiangensis in China.

Here, we report the complete genome sequence of Erwinia amylovora strain 99east-3-1, which was isolated from Pyrus sinkiangensis in Xinjiang Uygur Autonomous Region, China.

ntrC Contributes to Nitrogen Utilization, Stress Tolerance, and Virulence in Acidovorax citrulli.

Bacterial fruit blotch (BFB), caused by Acidovorax citrulli, severely damages watermelon, melon, and other cucurbit crops worldwide. Nitrogen, one of the most important limiting elements in the environment, is necessary for the growth and reproduction of bacteria. As a nitrogen-regulating gene, ntrC plays an important role in maintaining bacterial nitrogen utilization and biological nitrogen fixation. However, the role of ntrC has not been determined for A. citrulli. In this study, we constructed a ntrC deletion mutant and a corresponding complementary strain in the background of the A. citrulli wild-type strain, Aac5. Through phenotype assays and qRT-PCR analysis, we investigated the role of ntrC in A. citrulli in nitrogen utilization, stress tolerance, and virulence against watermelon seedlings. Our results showed that the A. citrulli Aac5 ntrC deletion mutant lost the ability to utilize nitrate. The ntrC mutant strain also exhibited significantly decreased virulence, in vitro growth, in vivo colonization ability, swimming motility, and twitching motility. In contrast, it displayed significantly enhanced biofilm formation and tolerance to stress induced by oxygen, high salt, and copper ions. The qRT-PCR results showed that the nitrate utilization gene nasS; the Type III secretion system-related genes hrpE, hrpX, and hrcJ; and the pili-related gene pilA were significantly downregulated in the ntrC deletion mutant. The nitrate utilization gene nasT, and the flagellum-related genes flhD, flhC, fliA, and fliC were significantly upregulated in the ntrC deletion mutant. The expression levels of ntrC gene in the MMX-q and XVM2 media were significantly higher than in the KB medium. These results suggest that the ntrC gene plays a pivotal role in the nitrogen utilization, stress tolerance, and virulence of A. citrulli.

Draft Genome Sequence of a Xylella fastidiosa Strain Causing Bacterial Leaf Scorch of American Elm in Washington, DC.

Here, we report the draft genome sequence of Xylella fastidiosa strain ATCC 35873, which was obtained from the American Type Culture Collection and was originally isolated from a symptomatic American elm tree grown in Washington, DC. The ATCC 35873 genome contains 2,454,216 bp and has a GC content of 51.68%.

Identification of the Causal Agent of Bacterial Leaf Spot on Watermelon in China.

Watermelon diseases caused by pathogenic bacteria were endemic in Liaoning and Jilin Provinces from 2019 to 2020 in China, resulting in serious economic losses to the watermelon industry. This study characterized 56 strains isolated from symptomatic watermelon leaves collected from Liaoning and Jilin Provinces. Through morphological observation, 16S rRNA and gyrB sequence analysis, and BIOLOG profiles, the pathogen was identified as Pseudomonas syringae. In China, the watermelon disease caused by P. syringae was reported for the first time. The multilocus sequence analysis showed that the isolated strains belonged to three different clades within P. syringae phylogroup 2. Interestingly, most of them (79%) belonged to clade 2a, 14% were clade 2b, and 7% were clade 2d. This indicates that bacterial leaf spot outbreaks of watermelon in China were caused by multiple sources and mainly by P. syringae clade 2a.

A putative multi-sensor hybrid histidine kinase, BarA Ac , inhibits the expression of the type III secretion system regulator HrpG in Acidovorax citrulli.

Bacterial fruit blotch (BFB), caused by Acidovorax citrulli, severely damages watermelon, melon, and other cucurbit crops worldwide. Although many virulence determinants have been identified in A. citrulli, including swimming motility, twitching motility, biofilm formation, and the type III secretion system (T3SS), research on their regulation is lacking. To study virulence regulation mechanisms, we found a putative histidine kinase BarA Ac that may be related to the T3SS regulator HrpG in A. citrulli. We deleted and characterized barAAc (Aave_2063) in A. citrulli Aac5 strain. Compared to the wild-type Aac5, virulence and early proliferation of barAAc mutant in host watermelon cotyledons were significantly increased, and induction of hypersensitive response in non-host tobacco was accelerated, while biofilm formation and swimming motility were significantly reduced. In addition, the transcriptomic analysis revealed that the expression of many T3SS-related genes was upregulated in the ΔbarAAc deletion mutant when cultured in KB medium. Meanwhile, the ΔbarAAc deletion mutant showed increased accumulation of the T3SS regulator HrpG in KB medium, which may account for the increased deployment of T3SS. This suggests that the putative histidine kinase BarA Ac is able to repress the T3SS expression by inhibiting HrpG in the KB medium, which appears to be important for rational energy allocation. In summary, our research provides further understanding of the regulatory network of A. citrulli virulence.

Acidovorax citrulli Effector AopV Suppresses Plant Immunity and Interacts with Aromatic Dehydratase ADT6 in Watermelon.

Bacterial fruit blotch (BFB) is a disease of cucurbit plants caused by Acidovorax citrulli. Although A. citrulli has great destructive potential, the molecular mechanisms of pathogenicity of A. citrulli are not clear, particularly with regard to its type III secreted effectors. In this study, we characterized the type III secreted effector protein, AopV, from A. citrulli strain Aac5. We show that AopV significantly inhibits reactive oxygen species and the expression of PTI marker genes, and helps the growth of Pseudomonas syringae D36E in Nicotiana benthamiana. In addition, we found that the aromatic dehydratase ADT6 from watermelon was a target of AopV. AopV interacts with ADT6 in vivo and in vitro. Subcellular localization indicated ADT6 and AopV were co-located at the cell membrane. Together, our results reveal that AopV suppresses plant immunity and targets ADT6 in the cell membrane. These findings provide an new characterization of the molecular interaction of A. citrulli effector protein AopV with host cells.

Hcp of the Type VI Secretion System (T6SS) in Acidovorax citrulli Group II Strain Aac5 Has a Dual Role as a Core Structural Protein and an Effector Protein in Colonization, Growth Ability, Competition, Biofilm Formation, and Ferric Iron Absorption.

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.

Essential Acidovorax citrulli Virulence Gene hrpE Activates Host Immune Response against Pathogen.

Bacterial fruit blotch (BFB) caused by Acidovorax citrulli (Ac) is a devastating watermelon disease that severely impacts the global watermelon industry. Like other Gram-negative bacteria, the type three secretion system (T3SS) is the main pathogenicity factor of A. citrulli. The T3SS apparatus gene hrpE codes for the Hrp pilus and serves as a conduit to secret effector proteins into host cells. In this study, we found that the deletion of hrpE in A. citrulli results in the loss of pathogenicity on hosts and the hypersensitive response on non-hosts. In addition, the A. citrulli hrpE mutant showed a reduction in in vitro growth, in planta colonization, swimming and twitching motility, and displayed increases in biofilm formation ability compared to the wild type. However, when HrpE was transiently expressed in hosts, the defense responses, including reactive oxygen species bursts, callose deposition, and expression of defense-related genes, were activated. Thus, the A. Citrulli growth in HrpE-pretreated hosts was suppressed. These results indicated that HrpE is essential for A. citrulli virulence but can also be used by hosts to help resist A. citrulli. Our findings provide a better understanding of the T3SS pathogenesis in A. citrulli, thus providing a molecular basis for biopesticide development, and facilitating the effective control of BFB.

Transcriptomic and Functional Analyses Reveal Roles of AclR, a luxR-type Global Regular, in Regulating Motility and Virulence of Acidovorax citrulli.

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.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Acidovorax citrulli Type III Effector AopP Suppresses Plant Immunity by Targeting the Watermelon Transcription Factor WRKY6.

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.

Identification and Functional Analysis of AopN, an Acidovorax Citrulli Effector that Induces Programmed Cell Death in Plants.

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.

A LuxR-type regulator, AcrR, regulates flagellar assembly and contributes to virulence, motility, biofilm formation, and growth ability of Acidovorax citrulli.

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.

Involvement of hrpX and hrpG in the Virulence of Acidovorax citrulli Strain Aac5, Causal Agent of Bacterial Fruit Blotch in Cucurbits.

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.