Mutational analysis of predicted active site residues of an exoglucanase secreted by Xanthomonas oryzae pv. oryzae to determine their role in catalysis and in virulence on rice.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight disease in rice. As a part of its virulence repertoire, Xoo secretes a cell wall degrading enzyme Cellobiosidase (CbsA), which is a critical virulence factor and also a determinant of tissue specificity. CbsA protein is made up of an N-terminal catalytic domain and a C-terminal fibronectin type III domain. According to the CAZy classification, the catalytic domain of CbsA protein belongs to the glycosyl hydrolase-6 (GH6) family that performs acid-base catalysis. However, the identity of the catalytic acid and the catalytic base of CbsA is not known. Based on the available structural and biochemical data, we identified putative catalytic residues and probed them by site-directed mutagenesis. Intriguingly, the biochemical analysis showed that none of the mutations abolishes the catalytic activity of CbsA, an observation that is contrary to other GH6 family members. All the mutants exhibited altered enzymatic activity and caused significant virulence deficiency in Xoo emphasising the requirement of specific exoglucanase activity of wild-type CbsA for virulence on rice. Our study highlights the need for further studies and the detailed characterisation of bacterial exoglucanases.

Xanthomonas oryzae pv. oryzae Exoribonuclease R Is Required for Complete Virulence in Rice, Optimal Motility, and Growth Under Stress.

Exoribonuclease R (RNase R) is a 3' hydrolytic exoribonuclease that can degrade structured RNA. Mutation in RNase R affects virulence of certain human pathogenic bacteria. The aim of this study was to determine whether RNase R is necessary for virulence of the phytopathogen that causes bacterial blight in rice, Xanthomonas oryzae pv. oryzae (Xoo). In silico analysis has indicated that RNase R is highly conserved among various xanthomonads. Amino acid sequence alignment of Xoo RNase R with RNase R from various taxa indicated that Xoo RNase R clustered with RNase R of order Xanthomonadales. To study its role in virulence, we generated a gene disruption mutant of Xoo RNase R. The Xoo rnr- mutant is moderately virulence deficient, and the complementing strain (rnr-/pHM1::rnr) rescued the virulence deficiency of the mutant. We investigated swimming and swarming motilities in both nutrient-deficient minimal media and nutrient-optimal media. We observed that RNase R mutation has adversely affected the swimming and swarming motilities of Xoo in optimal media. However, in nutrient-deficient media only swimming motility was noticeably affected. Growth curves in optimal media at suboptimal temperature (15°C cold stress) indicate that the Xoo rnr- mutant grows more slowly than the Xoo wild type and complementing strain (rnr-/pHM1::rnr). Given these findings, we report for the first time that RNase R function is necessary for complete virulence of Xoo in rice. It is also important for motility of Xoo in media and for growth of Xoo at suboptimal temperature.

DEAD box helicases as promising molecular tools for engineering abiotic stress tolerance in plants.

Diverse abiotic stresses constitute one of the major factors which adversely affect the normal plant growth and development which results worldwide in decreased agricultural productivity. At present, utilization of new molecular tools to achieve improved stress tolerance and increased crop productivity is highly desirable. Abiotic stress in plants induces expression of a wide range of genes like transcription factors, defense related genes and so on, and the products of these genes are important in combating stress conditions. Helicases are one such category of proteins that play a key role in maintaining the genomic integrity of the cell by participating in nucleic acid mediated processes such as recombination, replication, and repair of DNA as well as the unwinding of misfolded RNA structures that are formed during stress conditions. The DEAD box helicases are a subgroup of helicases which contain the amino acids Asp-Glu-Ala-Asp (DEAD) and are involved in the above molecular functions that mediate adaptation to stress. Overexpression of DEAD box helicases is known to provide stress tolerance in various plants and thus their use in developing stress tolerant plants is gaining importance. The plausible physiological mechanisms of helicases in bestowing abiotic stress tolerance of plants include ROS scavenging, enhanced photosynthesis, ion homeostasis and regulation of various stress responsive genes. In this review, the characteristics of plant DEAD box helicases and the stress conditions under which they express are discussed. We have provided a detailed description on the transgenic plants overexpressing DEAD box helicases with an emphasis on their stress tolerance abilities.

A mutation in an exoglucanase of Xanthomonas oryzae pv. oryzae, which confers an endo mode of activity, affects bacterial virulence, but not the induction of immune responses, in rice.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, a serious disease of rice. Xoo secretes a repertoire of cell wall-degrading enzymes, including cellulases, xylanases and pectinases, to degrade various polysaccharide components of the rice cell wall. A secreted Xoo cellulase, CbsA, is not only a key virulence factor of Xoo, but is also a potent inducer of innate immune responses of rice. In this study, we solved the crystal structure of the catalytic domain of the CbsA protein to a resolution of 1.86 Å. The core structure of CbsA shows a central distorted TIM barrel made up of eight ß strands with N- and C-terminal loops enclosing the active site, which is a characteristic structural feature of an exoglucanase. The aspartic acid at the 131st position of CbsA was predicted to be important for catalysis and was therefore mutated to alanine to study its role in the catalysis and biological functions of CbsA. Intriguingly, the D131A CbsA mutant protein displayed the enzymatic activity of a typical endoglucanase. D131A CbsA was as proficient as wild-type (Wt) CbsA in inducing rice immune responses, but was deficient in virulence-promoting activity. This indicates that the specific exoglucanase activity of the Wt CbsA protein is required for this protein to promote the growth of Xoo in rice.

Identification of Pectin Degrading Enzymes Secreted by Xanthomonas oryzae pv. oryzae and Determination of Their Role in Virulence on Rice.

Xanthomonas oryzae pv.oryzae (Xoo) causes the serious bacterial blight disease of rice. Xoo secretes a repertoire of plant cell wall degrading enzymes (CWDEs) like cellulases, xylanases, esterases etc., which act on various components of the rice cell wall. The major cellulases and xylanases secreted by Xoo have been identified and their role in virulence has been determined. In this study, we have identified some of the pectin degrading enzymes of Xoo and assessed their role in virulence. Bioinformatics analysis indicated the presence of four pectin homogalacturonan (HG) degrading genes in the genome of Xoo. The four HG degrading genes include one polygalacturonase (pglA), one pectin methyl esterase (pmt) and two pectate lyases (pel and pelL). There was no difference in the expression of pglA, pmt and pel genes by laboratory wild type Xoo strain (BXO43) grown in either nutrient rich PS medium or in plant mimic XOM2 medium whereas the expression of pelL gene was induced in XOM2 medium as indicated by qRT-PCR experiments. Gene disruption mutations were generated in each of these four genes. The polygalacturonase mutant pglA- was completely deficient in degrading the substrate Na-polygalacturonicacid (PGA). Strains carrying mutations in the pmt, pel and pelL genes were as efficient as wild type Xoo (BXO43) in cleaving PGA. These observations clearly indicate that PglA is the major pectin degrading enzyme produced by Xoo. The pectin methyl esterase, Pmt, is the pectin de-esterifying enzyme secreted by Xoo as evident from the enzymatic activity assay performed using pectin as the substrate. Mutations in the pglA, pmt, pel and pelL genes have minimal effects on virulence. This suggests that, as compared to cellulases and xylanases, the HG degrading enzymes may not have a major role in the pathogenicity of Xoo.

Action of Multiple Cell Wall-Degrading Enzymes Is Required for Elicitation of Innate Immune Responses During Xanthomonas oryzae pv. oryzae Infection in Rice.

Xanthomonas oryzae pv. oryzae secretes a number of plant cell wall-degrading enzymes (CWDEs) whose purified preparations induce defense responses in rice. These defense responses are suppressed by X. oryzae pv. oryzae using type 3 secretion system (T3SS) effectors and a type 3 secretion system mutant (T3SS(-)) of X. oryzae pv. oryzae is an inducer of rice defense responses. We assessed the role of individual CWDEs in induction of rice defense responses during infection, by mutating them in the genetic background of a T3SS(-). We mutated the genes for five different plant CWDEs secreted by X. oryzae pv. oryzae, including two cellulases (clsA and cbsA), one xylanase (xyn), one pectinase (pglA), and an esterase (lipA), singly in a T3SS(-) background. We have demonstrated that, as compared with a T3SS(-) of X. oryzae pv. oryzae, a cbsA(-)T3SS(-), a clsA(-)T3SS(-), and a xyn(-)T3SS(-) are deficient in induction of rice immune responses such as callose deposits and programmed cell death. In comparison, a lipA(-) T3SS(-) and a pglA(-)T3SS(-) is as efficient in induction of host defense responses as a T3SS(-). Overall, these results indicate that the collective action of X. oryzae pv. oryzae-secreted ClsA, CbsA, and Xyn proteins is required for induction of rice defense responses during infection.