Dissection of Quorum-Sensing Genes in Burkholderia glumae Reveals Non-Canonical Regulation and the New Regulatory Gene tofM for Toxoflavin Production

Burkholderia glumae causes bacterial panicle blight of rice and produces major virulence factors, including toxoflavin, under the control of the quorum-sensing (QS) system mediated by the luxI homolog, tofI, and the luxR homolog, tofR. In this study, a series of markerless deletion mutants of B. glumae for tofI and tofR were generated using the suicide vector system, pKKSacB, for comprehensive characterization of the QS system of this pathogen. Consistent with the previous studies by other research groups, ΔtofI and ΔtofR strains of B. glumae did not produce toxoflavin in Luria-Bertani (LB) broth. However, these mutants produced high levels of toxoflavin when grown in a highly dense bacterial inoculum (∼ 10¹¹ CFU/ml) on solid media, including LB agar and King’s B (KB) agar media. The ΔtofI/ΔtofR strain of B. glumae, LSUPB201, also produced toxoflavin on LB agar medium. These results indicate the presence of previously unknown regulatory pathways for the production of toxoflavin that are independent of tofI and/or tofR. Notably, the conserved open reading frame (locus tag: bglu_2g14480) located in the intergenic region between tofI and tofR was found to be essential for the production of toxoflavin by tofI and tofR mutants on solid media. This novel regulatory factor of B. glumae was named tofM after its homolog, rsaM, which was recently identified as a novel negative regulatory gene for the QS system of another rice pathogenic bacterium, Pseudomonas fuscovaginae. The ΔtofM strain of B. glumae, LSUPB286, produced a less amount of toxoflavin and showed attenuated virulence when compared with its wild type parental strain, 336gr-1, suggesting that tofM plays a positive role in toxoflavin production and virulence. In addition, the observed growth defect of the ΔtofI strain, LSUPB145, was restored by 1 µM N-octanoyl homoserine lactone (C8-HSL).     

Differential regulation of toxoflavin production and its role in the enhanced virulence of Burkholderia gladioli

Burkholderia gladioli is a causal agent of bacterial panicle blight and sheath/grain browning in rice in many countries. Many strains produce the yellow pigment toxoflavin, which is highly toxic to plants, fungi, animals and microorganisms. Although there have been several studies on the toxoflavin biosynthesis system of B. glumae, it is still unclear how B. gladioli activates toxoflavin biosynthesis. In this study, we explored the genomic organization of the toxoflavin system of B. gladioli and its biological functions using comparative genomic analysis between toxoflavin‐producing strains (B. glumae BGR1 and B. gladioli BSR3) and a strain not producing toxoflavin (B. gladioli KACC11889). The latter exhibits normal physiological characteristics similar to other B. gladioli strains. Burkholderia gladioli KACC11889 possesses all the genes involved in toxoflavin biosynthesis, but lacks the quorum‐sensing (QS) system that functions as an on/off switch for toxoflavin biosynthesis. These data suggest that B. gladioli has evolved to use the QS signalling cascade of toxoflavin production (TofI/TofR of QS → ToxJ or ToxR → tox operons) similar to that in B. glumae. However, some strains may have evolved to eliminate toxoflavin production through deletion of the QS genes. In addition, we demonstrate that the toxoflavin biosynthetic system enhances the virulence of B. gladioli. These findings provide another line of evidence supporting the differential regulation of the toxoflavin system in Burkholderia strains.     

The roles of the shikimate pathway genes,aroA and aroB,in virulence,growth and UV tolerance of Burkholderia glumae strain 411gr‐6

Burkholderia glumae is the major causal agent of bacterial panicle blight of rice, which is a growing disease problem for rice growers worldwide. In our previous study, some B. glumae strains showed pigmentation phenotypes producing at least two (yellow–green and purple) pigment compounds in casein–peptone–glucose agar medium. The B. glumae strains LSUPB114 and LSUPB116 are pigment‐deficient mutant derivatives of the virulent and pigment‐proficient strain 411gr‐6, having mini‐Tn5gus insertions in aroA encoding 3‐phosphoshikimate 1‐carboxyvinyltransferase and aroB encoding 3‐dehydroquinate synthase, respectively. Both enzymes are known to be involved in the shikimate pathway, which leads to the synthesis of aromatic amino acids. Here, we demonstrate that aroA and aroB are required for normal virulence in rice and onion, growth in M9 minimal medium and tolerance to UV light, but are dispensable for the production of the phytotoxin toxoflavin. These results suggest that the shikimate pathway is involved in bacterial pathogenesis by B. glumae without a significant role in the production of toxoflavin, a major virulence factor of this pathogen.     

Involvement of a Quorum-Sensing-Regulated Lipase Secreted by a Clinical Isolate of Burkholderia glumae in Severe Disease Symptoms in Rice

Burkholderia glumae is an emerging rice pathogen in several areas around the world. Closely related Burkholderia species are important opportunistic human pathogens for specific groups of patients, such as patients with cystic fibrosis and patients with chronic granulomatous disease. Here we report that the first clinical isolate of B. glumae, strain AU6208, has retained its capability to be very pathogenic to rice. As previously reported for rice isolate B. glumae BGR1 (and also for the clinical isolate AU6208), TofI or TofR acyl homoserine lactone (AHL) quorum sensing played a pivotal role in rice virulence. We report that AHL quorum sensing in B. glumae AU6208 regulates secreted LipA lipase and toxoflavin, the phytotoxin produced by B. glumae. B. glumae AU6208 lipA mutants were no longer pathogenic to rice, indicating that the lipase is an important virulence factor. It was also established that type strain B. glumae ATCC 33617 did not produce toxoflavin and lipase and was nonpathogenic to rice. It was determined that in strain ATCC 33617 the LuxR family quorum-sensing sensor/regulator TofR was inactive. Introducing the tofR gene of B. glumae AU6208 in strain ATCC 33617 restored its ability to produce toxoflavin and the LipA lipase. This study extends the role of AHL quorum sensing in rice pathogenicity through the regulation of a lipase which was demonstrated to be a virulence factor. It is the first report of a clinical B. glumae isolate retaining strong rice pathogenicity and finally determined that B. glumae can undergo phenotypic conversion through a spontaneous mutation in the tofR regulator.     

Type VI secretion systems of plant-pathogenic Burkholderia glumae BGR1 play a functionally distinct role in interspecies interactions and virulence

In the environment, bacteria show close association, such as interspecies interaction, with other bacteria as well as host organisms. The type VI secretion system (T6SS) in gram-negative bacteria is involved in bacterial competition or virulence. The plant pathogen Burkholderia glumae BGR1, causing bacterial panicle blight in rice, has four T6SS gene clusters. The presence of at least one T6SS gene cluster in an organism indicates its distinct role, like in the bacterial and eukaryotic cell targeting system. In this study, deletion mutants targeting four tssD genes, which encode the main component of T6SS needle formation, were constructed to functionally dissect the four T6SSs in B. glumae BGR1. We found that both T6SS group_4 and group_5, belonging to the eukaryotic targeting system, act independently as bacterial virulence factors toward host plants. In contrast, T6SS group_1 is involved in bacterial competition by exerting antibacterial effects. The ΔtssD1 mutant lost the antibacterial effect of T6SS group_1. The ΔtssD1 mutant showed similar virulence as the wild-type BGR1 in rice because the ΔtssD1 mutant, like the wild-type BGR1, still has key virulence factors such as toxin production towards rice. However, metagenomic analysis showed different bacterial communities in rice infected with the ΔtssD1 mutant compared to wild-type BGR1. In particular, the T6SS group_1 controls endophytic plant-associated bacteria such as Luteibacter and Dyella in rice plants and may have an advantage in competing with endophytic plant-associated bacteria for settlement inside rice plants in the environment. Thus, B. glumae BGR1 causes disease using T6SSs with functionally distinct roles.     

Infection Process of Burkholderia glumae in Rice Spikelets

Burkholderia glumae, which causes bacterial panicle blight of rice (BPBR), is a well‐known pathogen. The pathogen‐induced symptoms include seedling rot, grain rot and leaf‐sheath browning in rice plants. B. glumae can incubate in rice plants as endophytes before booting stage of rice. In this study, we constructed a gfp‐labelled system of B. glumae LMG 2196 and used SEM to clarify the colonization course of B. glumae at the heading stage. New locations of B. glumae were found. The pathogens initially distributed on the surface of the glumes and colonized in the glume hairs and cells of the edge of sterile lemma, palea and lemma. The base of glume hairs was the initial position for colonization. Bacterial population raised around glume hairs, penetrated into the inner surface of the palea and lemma, and spread on the gynoecium and stamens through contact. The spreading of B. glumae among the panicles mainly occurred through the contact or friction among glumes or leaf sheaths, but the inner spread of the stamens mainly occurred through the connective tissue of anther. We also detected the differences of bacterial content in stamens, gynoecia and glumes. The growing stage of B. glumae in spikelets could be divided into two sections. The biomass of all parts continued to increase to nearly 10⁸ CFU/g at 10 DAI. This caused wilt symptoms and stopped the pollination. This work showed that glume hairs played an important role in the initial colonization of B. glumae, and provides a foundation for further studies of the infection manner of B. glumae and other pathogenic bacteria.     

Quorum Sensing Controls Swarming Motility of Burkholderia glumae through Regulation of Rhamnolipids

Burkholderia glumae is a plant pathogenic bacterium that uses an acyl-homoserine lactone-mediated quorum sensing system to regulate protein secretion, oxalate production and major virulence determinants such as toxoflavin and flagella. B. glumae also releases surface-active rhamnolipids. In Pseudomonas aeruginosa and Burkholderia thailandensis, rhamnolipids, along with flagella, are required for the social behavior called swarming motility. In the present study, we demonstrate that quorum sensing positively regulates the production of rhamnolipids in B. glumae and that rhamnolipids are necessary for swarming motility also in this species. We show that a rhlA- mutant, which is unable to produce rhamnolipids, loses its ability to swarm, and that this can be complemented by providing exogenous rhamnolipids. Impaired rhamnolipid production in a quorum sensing-deficient B. glumae mutant is the main factor responsible for its defective swarming motility behaviour.     

Diversities in Virulence,Antifungal Activity,Pigmentation and DNA Fingerprint among Strains of Burkholderia glumae

Burkholderia glumae is the primary causal agent of bacterial panicle blight of rice. In this study, 11 naturally avirulent and nine virulent strains of B. glumae native to the southern United States were characterized in terms of virulence in rice and onion, toxofalvin production, antifungal activity, pigmentation and genomic structure. Virulence of B. glumae strains on rice panicles was highly correlated to virulence on onion bulb scales, suggesting that onion bulb can be a convenient alternative host system to efficiently determine the virulence of B. glumae strains. Production of toxoflavin, the phytotoxin that functions as a major virulence factor, was closely associated with the virulence phenotypes of B. glumae strains in rice. Some strains of B. glumae showed various levels of antifungal activity against Rhizoctonia solani, the causal agent of sheath blight, and pigmentation phenotypes on casamino acid-peptone-glucose (CPG) agar plates regardless of their virulence traits. Purple and yellow-green pigments were partially purified from a pigmenting strain of B. glumae, 411gr-6, and the purple pigment fraction showed a strong antifungal activity against Collectotrichum orbiculare. Genetic variations were detected among the B. glumae strains from DNA fingerprinting analyses by repetitive element sequence-based PCR (rep-PCR) for BOX-A1R-based repetitive extragenic palindromic (BOX) or enterobacterial repetitive intergenic consensus (ERIC) sequences of bacteria; and close genetic relatedness among virulent but pigment-deficient strains were revealed by clustering analyses of DNA fingerprints from BOX-and ERIC-PCR.