Joseph Bigirimana

A LuxR homologue of Xanthomonas oryzae pv. oryzae is required for optimal rice virulence

SUMMARY In Gram-negative bacteria a typical quorum sensing (QS) system usually involves the production and response to acylated homoserine lactones (AHLs). An AHL QS system is most commonly mediated by a LuxI family AHL synthase and a LuxR family AHL response regulator. This study reports for the first time the presence of a LuxR family-type regulator in Xanthomonas oryzae pv. oryzae (Xoo), which has been designated as OryR. The primary structure of OryR contains the typical signature domains of AHL QS LuxR family response regulators: an AHL-binding and a HTH DNA binding motif. The oryR gene is conserved among 26 Xoo strains and is also present in the genomes of close relatives X. campestris pv. campestris and X. axonopodis pv. citri. Disrupting oryR in three Xoo strains resulted in a significant reduction of rice virulence. The wild-type Xoo strains do not seem to produce AHLs and analysis of the Xoo sequenced genomes did not reveal the presence of a LuxI-family AHL synthase. The OryR protein was shown to be induced by macerated rice and affected the production of two secreted proteins: a cell-wall-degrading cellobiosidase and a 20-kDa protein of unknown function. By expressing and purifying OryR it was then observed that it was solubilized when grown in the presence of rice extract indicating that there could be a molecule(s) in rice which binds OryR. The role of OryR as a possible in planta induced LuxR family regulator is discussed.

Involvement of a quorum-sensing-regulated lipase secreted by a clinical isolate of Burkholderia glumae in severe disease symptoms in rice.

ABSTRACT 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.

The plant pathogen Pseudomonas fuscovaginae contains two conserved quorum sensing systems involved in virulence and negatively regulated by RsaL and the novel regulator RsaM.

Pseudomonas fuscovaginae is a Gram-negative fluorescent pseudomonad pathogenic towards several plant species. Despite its importance as a plant pathogen, no molecular studies of virulence have thus far been reported. In this study we show that P. fuscovaginae possesses two conserved N-acyl homoserine lactone (AHL) quorum sensing (QS) systems which we designated PfsI/R and PfvI/R. The PfsI/R system is homologous to the BviI/R system of Burkholderia vietnamiensis and produces and responds to C10-HSL and C12-HSL whereas PfvI/R is homologous to the LasI/R system of Pseudomonas aeruginosa and produces several long-chain 3-oxo-HSLs and responds to 3-oxo-C10-HSL and 3-oxo-C12-HSL and at high AHL concentrations can also respond to structurally different long-chain AHLs. Both systems were found to be negatively regulated by a repressor protein which was encoded by a gene located intergenically between the AHL synthase and LuxR-family response regulator. The pfsI/R system was regulated by a novel repressor designated RsaM while the pfvI/R system was regulated by both the RsaL repressor and by RsaM. The two systems are not transcriptionally hierarchically organized but share a common AHL response and both are required for plant virulence. Pseudomonas fuscovaginae has therefore a unique complex regulatory network composed of at least two different repressors which directly regulate the AHL QS systems and pathogenicity.

The presence, type and role of N-acyl homoserine lactone quorum sensing in fluorescent Pseudomonas originally isolated from rice rhizospheres are unpredictable

In Gram-negative bacteria, a typical quorum-sensing (QS) system involves the production and response to N-acyl homoserine lactones (AHLs). It still remains unclear as to how pivotal and conserved AHL QS is in root-colonizing rhizosphere Pseudomonas. We, therefore, performed a systematic study of AHL QS on a set of 50 rice rhizosphere Pseudomonas isolates. We also isolated the AHL QS genes in two representative strains and analyzed the role of AHL QS regulation of various phenotypes. Our results are discussed with the current knowledge of AHL QS of rhizosphere Pseudomonas, implicating a lack of conservation and an unpredictable role played by AHL QS in this group of bacteria.

Xanthomonas oryzae pv. oryzae XKK.12 Contains an AroQγ Chorismate Mutase That Is Involved in Rice Virulence

Chorismate mutase (CM) is a key enzyme in the shikimate pathway which is responsible for the synthesis of aromatic amino acids. There are two classes of CMs, AroQ and AroH, and several pathogenic bacteria have been reported to possess a subgroup of CMs designated AroQ(gamma). These CMs are usually exported to the periplasm or outside the cell; in a few cases, they have been reported to be involved in virulence and their precise role is currently unknown. Here, we report that the important rice pathogen Xanthomonas oryzae pv. oryzae XKK.12 produces an AroQ(gamma) CM which we have purified and characterized from spent supernatants. This enzyme is synthesized in planta and X. oryzae pv. oryzae knock-out mutants are hypervirulent to rice. The role of this enzyme in X. oryzae pv. oryzae rice virulence is discussed.

Bean Anthracnose: Virulence of Colletotrichum lindemuthianum Isolates from Burundi, Central Africa

The diversity of Colletotrichum lindemuthianum is a major limiting factor in control of anthracnose on bean (Phaseolus vulgaris), and race characterization of this pathogen is an important tool in breeding programs. Race characterization has been carried out on isolates from North, Central, and South America; Europe; and Asia, but little or no information exists on the diversity of C. lindemuthianum in Africa. In this work, 12 isolates from the major bean-growing areas of Burundi, Central Africa, were characterized. Their virulence was tested on 12 bean differential cultivars (1) and on 4 bean cultivars commonly grown in Burundi: 2 from local germ plasm (Muyinga-1 and Urubonobono) and 2 from Colombia (A 321 and Calima). Detached unifoliate bean leaves from 8-day-old plants were placed on a humid surface in trays and sprayed until runoff with a suspension of 106 spores ml-1. Trays covered with transparent plastic sheets to keep a minimum relative humidity of 92% were incubated at 20°C. Seven days after inoculation, symptoms were evaluated for severity on a scale of 1 to 9. Leaves scored as 1 to 3 were considered resistant. Races were characterized according to a numerical binary system (1). Nine races were identified: 9, 69, 87, 384, 385, 401, 448, 449, and 485. Seven of these races (9, 69, 87, 384, 401, 448, and 485) were described for the first time in Africa. Races 401 and 485 have not yet been reported in the literature. The most susceptible differential cultivars were Michelite, PI 207262, To, and Mexico 222. Muyinga-1, Urubonobono, and A 321 were sensitive to nine, six, and five races, respectively. There is a high diversity of C. lindemuthianum in Burundi, and the local germ plasm tested is very susceptible to the characterized races. Breeding programs in Burundi should focus on lines and cultivars, such as Tu, AB 136, G 2333, and Calima, that are resistant to all the races characterized in this study. Reference: (1) M. A. Pastor-Corrales. Phytopathology 81:694, 1991.