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.

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 β 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.

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.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight disease of rice. Xoo uses cell wall-degrading enzymes like Cellulase, Esterase (LipA), Cellobiosidase, Xylanase, etc. as virulence factors. As part of our on-going efforts in understanding the structural basis of these virulence factors, structure of LipA and CbsA (Catalytic domain) proteins were solved. LipA protein belongs to a new class of cell wall-degrading enzymes which has a unique mode of substrate recognition. CbsA protein has an N-terminal catalytic domain (Glycosyl hydrolase family-6) and a C-terminal fibronectin type 3 domain. Under laboratory conditions, Xoo-secreted CbsA is a truncated protein having just the catalytic domain. The structure reveals the presence of the catalytic tunnel being enclosed by two loops, characteristic of a typical exoglucanase. Based on the structure, key residues important for catalysis were predicted. These residues are D99, S105, D148, Y93 and D367. In Glycoside hydrolase family 6 there is general agreement as to the identity of the catalytic acid from both crystallographic and solution studies. In case of CbsA this residue is D148. Surprisingly, CbsA D148A mutant shows catalytic activity. To determine if the enzymatic activity is required for its ability to induce immune responses and for its role in virulence, biochemically inactive forms of CbsA which abrogate the enzymatic activity are being generated. The C-terminal region of CbsA is the fibronectin type 3 (FN3) domain. FN3 domain has been reported in bacterial carbohydrate active enzymes, where it is implicated in binding of sugar groups or promotes hydrolysis. However, the distinctive feature of carbohydrate binding module, such as a large aromatic platform, is not detected on the FN3 domain. Cell fractionation studies indicate that the FN3 domain of CbsA is cleaved off in the cytoplasm. CbsA protein without the FN3 domain is biochemically active and can induce immune response in rice tissues. To assess the role of the fibronectin domain in the function of CbsA, a deletion mutant of FN3 domain was generated in Xoo and this mutant was found to be deficient in virulence. Western blot analysis using anti-CbsA antibody indicated that the level of the secreted CbsA protein in the ΔFN3 mutant was around 95% less than the wild type. However, RT-PCR analysis showed equal levels of cbsA gene expression in ΔFN3 mutant and wild type. Taken together, these results suggest that the deletion of the fibronectin domain results in reduced level of secreted CbsA protein, either due to altered post-transcriptional processing or transport across the membrane.