yggS Encoding Pyridoxal 5′-Phosphate Binding Protein Is Required for Acidovorax citrulli Virulence

Bacterial fruit blotch, caused by seed-borne pathogen Acidovorax citrulli, poses a serious threat to the production of cucurbits globally. Although the disease can cause substantial economic losses, limited information is available about the molecular mechanisms of virulence. This study identified that, a random transposon insertion mutant impaired in the ability to elicit a hypersensitive response on tobacco. The disrupted gene in this mutant was determined to be Aave_0638, which is predicted to encode a YggS family pyridoxal phosphate-dependent enzyme. YggS is a highly conserved protein among multiple organisms, and is responsible for maintaining the homeostasis of pyridoxal 5′-phosphate and amino acids in cells. yggS deletion mutant of A. citrulli strain XjL12 displayed attenuated virulence, delayed hypersensitive response, less tolerance to H2O2 and pyridoxine, increased sensitivity to antibiotic β-chloro-D-alanine, and reduced swimming. In addition, RNA-Seq analysis demonstrated that yggS was involved in regulating the expression of certain pathogenicity-associated genes related to secretion, motility, quorum sensing and oxidative stress response. Importantly, YggS significantly affected type III secretion system and its effectors in vitro. Collectively, our results suggest that YggS is indispensable for A.citrulli virulence and expands the role of YggS in the biological processes.

Comparative Proteomic Analysis for a Putative Pyridoxal Phosphate-Dependent Aminotransferase Required for Virulence in Acidovorax citrulli

Acidovorax citrulli (Ac) is the causative agent of bacterial fruit blotch disease in watermelon. Since resistant cultivars have not yet been developed, the virulence factors/mechanisms of Ac need to be characterized. This study reports the functions of a putative pyridoxal phosphate-dependent aminotransferase (PpdaAc) that transfers amino groups to its substrates and uses pyridoxal phosphate as a coenzyme. It was observed that a ppdaAc knockout mutant had a significantly reduced virulence in watermelon when introduced via germinated-seed inoculation as well as leaf infiltration. Comparative proteomic analysis predicted the cellular mechanisms related to PpdaAc. Apart from causing virulence, the PpdaAc may have significant roles in energy production, cell membrane, motility, chemotaxis, post-translational modifications, and iron-related mechanisms. Therefore, it is postulated that PpdaAc may possess pleiotropic effects. These results provide new insights into the functions of a previously unidentified PpdaAc in Ac.

Identification of a Cupin Protein Gene Responsible for Pathogenicity, Phage Susceptibility and LPS Synthesis of Acidovorax citrulli

Bacteriophages infecting Acidovorax citrulli, the causal agent of bacterial fruit blotch, have been proven to be effective for the prevention and control of this disease. However, the occurrence of bacteriophage-resistant bacteria is one of hurdles in phage biocontrol and the understanding of phage resistance in this bacterium is an essential step. In this study, we aim to investigate possible phage resistance of A. citrulli and relationship between phage resistance and pathogenicity, and to isolate and characterize the genes involved in these phenomena. A phage-resistant and less-virulent mutant named as AC-17-G1 was isolated among 3,264 A. citrulli Tn5 mutants through serial spot assays and plaque assays followed by pathogenicity test using seed coating method. The mutant has the integrated Tn5 in the middle of a cupin protein gene. This mutant recovered its pathogenicity and phage sensitivity by complementation with corresponding wild-type gene. Site-directed mutation of this gene from wild-type by CRISPR/Cas9 system resulted in the loss of pathogenicity and acquisition of phage resistance. The growth of AC-17-G1 in King’s B medium was much less than the wild-type, but the growth turned into normal in the medium supplemented with D-mannose 6-phosphate or D-fructose 6-phosphate indicating the cupin protein functions as a phosphomannos isomerase. Sodium dodecyl sulfa analysis of lipopolysaccharide (LPS) extracted from the mutant was smaller than that from wild-type. All these data suggest that the cupin protein is a phosphomannos isomerase involved in LPS synthesis, and LPS is an important determinant of pathogenicity and phage susceptibility of A. citrulli.

Phenazine-1-carboxylic Acid Produced by Pseudomonas chlororaphis YL-1 Is Effective against Acidovorax citrulli

The bacterial pathogen Acidovorax citrulli causes the destructive fruit blotch (BFB) on cucurbit plants. Pseudomonas chlororaphis YL-1 is a bacterial strain isolated from Mississippi soil and its genome harbors some antimicrobial-related gene clusters, such as phenazine, pyrrolnitrin, and pyoverdine. Here, we evaluated the antimicrobial activity of strain YL-1 as compared with its deficient mutants of antimicrobial-related genes, which were obtained using a sacB-based site-specific mutagenesis strategy. We found that only phenazine-deficient mutants ΔphzE and ΔphzF almost lost the inhibitory effects against A. citrulli in LB plates compared with the wild-type strain YL-1, and that the main antibacterial compound produced by strain YL-1 in LB medium was phenazine-1-carboxylic acid (PCA) based on the liquid chromatography-mass spectrometry (LC-MS) analysis. Gene expression analyses revealed that PCA enhanced the accumulation of reactive oxygen species (ROS) and increased the activity of catalase (CAT) in A. citrulli. The inhibition effect of PCA against A. citrulli was lowered by adding exogenous CAT. PCA significantly upregulated the transcript level of katB from 6 to 10 h, which encodes CAT that helps to protect the bacteria against oxidative stress. Collectively, the findings of this research suggest PCA is one of the key antimicrobial metabolites of bacterial strain YL-1, a promising biocontrol agent for disease management of BFB of cucurbit plants.

Breaching the cell-envelope barriers of gram-positive and fungal microbes by a type VI secretion system in Acidovorax citrulli

The type VI secretion system (T6SS) is a double-tubular toxin-injection nanomachine widely found in gram-negative human and plant pathogens. The current model depicts that the T6SS spear-like Hcp tube is powered by the contraction of an outer sheath to drill through the envelope of a neighboring cell, achieving cytosol to cytosol delivery. However, gram-positive bacteria seem to be impenetrable to such T6SS action. Here we report that a plant pathogen Acidovorax citrulli (AC) deploys a highly potent T6SS to kill a range of bacteria including Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Mycobacterium smegmatis as well as fungal species including Candida albicans and Pichia pastoris. Using bioinformatic and biochemical assays, we identified a group of T6SS effectors and characterized one effector RhsB that is critical for interspecies interaction. We report that RhsB contains a conserved YD-repeat domain and a C-terminal nuclease domain. Toxicity of RhsB was neutralized by its downstream immunity proteins through direct interaction. RhsB was cleaved at the C-terminal end and a catalytic mutation within the internal aspartic protease abolished such cleavage. Collectively, the T6SS of AC displays potent activities to penetrate the cell envelope barriers of gram-positive and fungal species, highlighting the greatly expanded capabilities of T6SS in modulating microbiome compositions in complex environments.

Natural variation in a short region of the Acidovorax citrulli type III-secreted effector AopW1 is associated with differences in cytotoxicity

Bacterial fruit blotch (BFB) is a serious disease of melon and watermelon caused by the Gram-negative bacterium Acidovorax citrulli. The strains of the pathogen can be divided into two major genetic groups, I and II. While group I strains are strongly associated with melon, group II strains are more aggressive on watermelon. Like many pathogenic bacteria, A. citrulli secretes a variety of protein effectors to the host cell via the type III secretion system. In the present study, we characterized AopW1, an A. citrulli type III-secreted effector that shares similarity with the actin cytoskeleton-disrupting effector HopW1 of Pseudomonas syringae and with effectors from other plant-pathogenic bacterial species. aopW1 is present in group I and II strains, encoding products of 485 amino acids. Although highly conserved in most of the sequence, AopW1 has a highly variable region (HVR) within amino acid positions 147 to 192, including 14 amino acid differences between groups. Here we show that group I AopW1 is more toxic to yeast and plant cells than group II AopW1, having a stronger actin filament disruption activity, and increased ability to reduce plant callose deposition. We demonstrate the role of some of these 14 amino acid positions in determining the phenotypic differences between the two versions of the effector. Moreover, cellular analyses revealed that in addition to the interaction with actin filaments, AopW1 is localized to the endoplasmic reticulum, chloroplasts, and early and recycling plant endosomes, with differences observed between the two AopW1 versions. Finally, we show that overexpression of the endosome-associated protein EHD1 that increases cellular recycling, attenuates the toxic effects exerted by AopW1 and increases defence responses. This study provides insights into the HopW1 family of bacterial effectors and their interactions with the plant cell and provides first evidence on the involvement of EHD1 in response to biotic stress.

Napthoquinones from Neocosmospora sp.—Antibiotic Activity against Acidovorax citrulli, the Causative Agent of Bacterial Fruit Blotch in Watermelon and Melon

Bacterial fruit blotch (BFB) is a bacterial disease that devastates Cucurbitaceae crops worldwide, causing significant economic losses. Currently, there is no means to treat or control the disease. This study focused on exploring the antibacterial properties of endophytic fungi against Acidovorax citrulli (Aac), the causative agent of BFB. Based on disc diffusion, time kill and MIC microdilution broth assays, four endophytes showed promise in controlling Aac. Nonetheless, only one strain, Neocosmospora sp. MFLUCC 17-0253, reduced the severity of disease on watermelon and melon seedlings up to 80%. Structure analysis revealed production of several compounds by the fungus. Three of these secondary metabolites, including mixture of 2-methoxy-6-methyl-7-acetonyl-8-hydroxy-1,4-maphthalenedione and 5,8-dihydroxy-7-acetonyl-1,4-naphthalenedione, anhydrojavanicin, and fusarnaphthoquinones B exhibited antagonistic activity against Aac. The chemical profile data in planta experiment analyzed by LC-Q/TOF-MS suggested successful colonization of endophytic fungi in their host plant and different metabolic profiles between treated and untreated seedling. Biofilm assay also demonstrated that secondary metabolites of Neocosmospora sp. MFLUCC 17-0253 significantly inhibited biofilm development of Aac. To the best of our knowledge, secondary metabolites that provide significant growth inhibition of Aac are reported for the first time. Thus, Neocosmospora sp. MFLUCC 17-0253 possesses high potential as a biocontrol agent for BFB disease.