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.

BackgroundRice, a major food crop of the world, endures many major biotic stresses like bacterial blight (BB), fungal blast (BL) and the insect Asian rice gall midge (GM) that cause significant yield losses. Progress in tagging, mapping and cloning of several resistance (R) genes against aforesaid stresses has led to marker assisted multigene introgression into elite cultivars for multiple and durable resistance. However, no detailed study has been made on possible interactions among these genes when expressed simultaneously under combined stresses.ResultsOur studies monitored expression profiles of 14 defense related genes in 11 rice breeding lines derived from an elite cultivar with different combination of R genes against BB, BL and GM under single and multiple challenge. Four of the genes found implicated earlier under combined GM and BB stress were confirmed to be induced (≥ 2 fold) in stem tissue following GM infestation; while one of these, cytochrome P450 family protein, was also induced in leaf in plants challenged by either BB or BL but not together. Three of the genes highlighted earlier in plants challenged by both BB and BL were also found induced in stem under GM challenge. Pi54 the target R gene against BL was also found induced when challenged by GM. Though expression of some genes was noted to be inhibited under combined pest challenge, such effects did not result in compromise in resistance against any of the target pests.ConclusionWhile R genes generally tended to respond to specific pest challenge, several of the downstream defense genes responded to multiple pest challenge either single, sequential or simultaneous, without any distinct antagonism in expression of resistance to the target pests in two of the pyramided lines RPNF05 and RPNF08.Electronic supplementary materialThe online version of this article (10.1186/s12284-018-0231-4) contains supplementary material, which is available to authorized users.

Cellobiosidase (CbsA) is an important secreted virulence factor of Xanthomonas oryzae pv. oryzae (Xoo), which causes bacterial blight of rice. CbsA is one of several cell wall‐degrading enzymes secreted by Xoo via the type II secretion system (T2SS). CbsA is considered a fundamental virulence factor for vascular pathogenesis. CbsA has an N‐terminal glycosyl hydrolase domain and a C‐terminal fibronectin type III (FnIII) domain. Interestingly, the secreted form of CbsA lacks the FnIII domain during in planta growth. Here we show that the presence of the FnIII domain inhibits the enzyme activity of CbsA on polysaccharide substrates like carboxymethylcellulose. The FnIII domain is required for the interaction of CbsA with SecB chaperone, and this interaction is crucial for the stability and efficient transport of CbsA across the inner membrane. Deletion of the FnIII domain reduced virulence similar to ΔcbsA Xoo, which corroborates the importance of the FnIII domain in CbsA. Our work elucidates a hitherto unknown function of the FnIII domain in enabling the virulence‐promoting activity of CbsA.