Hitendra Kumar Patel

Cell Wall Degrading Enzyme Induced Rice Innate Immune Responses Are Suppressed by the Type 3 Secretion System Effectors XopN, XopQ, XopX and XopZ of Xanthomonas oryzae pv. oryzae

Innate immune responses are induced in plants and animals through perception of Damage Associated Molecular Patterns. These immune responses are suppressed by pathogens during infection. A number of studies have focussed on identifying functions of plant pathogenic bacteria that are involved in suppression of Pathogen Associated Molecular Pattern induced immune responses. In comparison, there is very little information on functions used by plant pathogens to suppress Damage Associated Molecular Pattern induced immune responses. Xanthomonas oryzae pv. oryzae , a gram negative bacterial pathogen of rice, secretes hydrolytic enzymes such as LipA (Lipase/Esterase) that damage rice cell walls and induce innate immune responses. Here, we show that Agrobacterium mediated transient transfer of the gene for XopN, a X . oryzae pv. oryzae type 3 secretion (T3S) system effector, results in suppression of rice innate immune responses induced by LipA. A xopN - mutant of X . oryzae pv. oryzae retains the ability to suppress these innate immune responses indicating the presence of other functionally redundant proteins. In transient transfer assays, we have assessed the ability of 15 other X . oryzae pv. oryzae T3S secreted effectors to suppress rice innate immune responses. Amongst these proteins, XopQ, XopX and XopZ are suppressors of LipA induced innate immune responses. A mutation in any one of the xopN, xopQ, xopX or xopZ genes causes partial virulence deficiency while a xopN - xopX - double mutant exhibits a greater virulence deficiency. A xopN - xopQ - xopX - xopZ - quadruple mutant of X . oryzae pv. oryzae induces callose deposition, an innate immune response, similar to a X . oryzae pv. oryzae T3S- mutant in rice leaves. Overall, these results indicate that multiple T3S secreted proteins of X . oryzae pv. oryzae can suppress cell wall damage induced rice innate immune responses.

The ColRS system of Xanthomonas oryzae pv. oryzae is required for virulence and growth in iron-limiting conditions

Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight of rice, produces siderophores only under iron-limiting conditions. We screened 15 400 mTn5-induced mutants of X. oryzae pv. oryzae and isolated 27 mutants that produced siderophores even under iron-replete conditions. We found that the mTn5 insertions in 25 of these mutants were in or close to six genes. Mutants with insertions in five of these genes [colS, XOO1806 (a conserved hypothetical protein), acnB, prpR and prpB] exhibited a deficiency for growth on iron-limiting medium and a decrease in virulence. Insertions in a sixth gene, XOO0007 (a conserved hypothetical protein), were found to affect the ability to grow on iron-limiting medium, but did not affect the virulence. Targeted gene disruptants for colR (encoding the predicted cognate regulatory protein for ColS) also exhibited a deficiency for growth on iron-limiting medium and a decrease in virulence. colR and colS mutants were defective in the elicitation of hypersensitive response symptoms on the nonhost tomato. In addition, colR and colS mutants induced a rice basal defence response, suggesting that they are compromised in the suppression of host innate immunity. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that a functional ColRS system is required for the optimal expression of several genes encoding components of the type 3 secretion system (T3SS) of X. oryzae pv. oryzae. Our results demonstrate the role of several novel genes, including colR/colS, in the promotion of growth on iron-limiting medium and the virulence of X. oryzae pv. oryzae.

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.

Rice Leaf Transcriptional Profiling Suggests a Functional Interplay Between Xanthomonas oryzae pv. oryzae Lipopolysaccharide and Extracellular Polysaccharide in Modulation of Defense Responses During Infection

Treatment of rice leaves with isolated Xanthomonas oryzae pv. oryzae lipopolysaccharide (LPS) induces the production of callose deposits, reactive oxygen species, and enhanced resistance against subsequent bacterial infection. Expression profiling of X. oryzae pv. oryzae LPS-treated rice (Oryza sativa subsp. indica) leaves showed that genes involved in the biosynthetic pathways for lignins, phenylpropanoids, chorismate, phenylalanine, salicylic acid, and ethylene, as well as a number of pathogenesis-related proteins are up-regulated. Gene ontology categories like cell-wall organization, defense response, stress response, and protein phosphorylation/kinases were found to be upregulated, while genes involved in photosynthesis were down-regulated. Coinfiltration with xanthan gum, the xanthomonas extracellular polysaccharide (EPS), suppressed LPS-induced callose deposition. Gene expression analysis of rice leaves that are treated with an EPS-deficient mutant of X. oryzae pv. oryzae indicated that a number of defense-regulated functions are up-regulated during infection. These transcriptional responses are attenuated in rice leaves treated with an EPS-deficient mutant that is also deficient in the O-antigen component of LPS. Overall, these results suggest that the O-antigen component of X. oryzae pv. oryzae LPS induces rice defense responses during infection and that these are suppressed by bacterial EPS.

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.

Identification of Loci of Pseudomonas syringae pv. actinidiae Involved in Lipolytic Activity and Their Role in Colonization of Kiwifruit Leaves

Bacterial canker disease caused by Pseudomonas syringae pv. actinidiae, an emerging pathogen of kiwifruit plants, has recently brought about major economic losses worldwide. Genetic studies on virulence functions of P. syringae pv. actinidiae have not yet been reported and there is little experimental data regarding bacterial genes involved in pathogenesis. In this study, we performed a genetic screen in order to identify transposon mutants altered in the lipolytic activity because it is known that mechanisms of regulation, production, and secretion of enzymes often play crucial roles in virulence of plant pathogens. We aimed to identify the set of secretion and global regulatory loci that control lipolytic activity and also play important roles in in planta fitness. Our screen for altered lipolytic activity phenotype identified a total of 58 Tn5 transposon mutants. Mapping all these Tn5 mutants revealed that the transposons were inserted in genes that play roles in cell division, chemotaxis, metabolism, movement, recombination, regulation, signal transduction, and transport as well as a few unknown functions. Several of these identified P. syringae pv. actinidiae Tn5 mutants, notably the functions affected in phosphomannomutase AlgC, lipid A biosynthesis acyltransferase, glutamate-cysteine ligase, and the type IV pilus protein PilI, were also found affected in in planta survival and/or growth in kiwifruit plants. The results of the genetic screen and identification of novel loci involved in in planta fitness of P. syringae pv. actinidiae are presented and discussed.

Expression Profile of Defense Genes in Rice Lines Pyramided with Resistance Genes Against Bacterial Blight, Fungal Blast and Insect Gall Midge

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.

A mutation in an exoglucanase of Xanthomonas oryzae pv. oryzae that 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 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.

Overexpression of a cell wall damage induced transcription factor, OsWRKY42, leads to enhanced callose deposition and tolerance to salt stress but does not enhance tolerance to bacterial infection

BackgroundMembers of the WRKY gene family play important roles in regulating plant responses to abiotic and biotic stresses. Treatment with either one of the two different cell wall degrading enzymes (CWDEs), LipaseA and CellulaseA, induces immune responses and enhances the expression of OsWRKY42 in rice. However, the role of OsWRKY42 in CWDE induced immune responses is not known.ResultsExpression of the rice transcription factor OsWRKY42 is induced upon treatment of rice leaves with CWDEs, wounding and salt. Overexpression of OsWRKY42 leads to enhanced callose deposition in rice and Arabidopsis but this does not enhance tolerance to bacterial infection. Upon treatment with NaCl, Arabidopsis transgenic plants expressing OsWRKY42 exhibited high levels of anthocyanin and displayed enhanced tolerance to salt stress. Treatment with either cellulase or salt induced the expression of several genes involved in JA biosynthesis and response in Arabidopsis. Ectopic expression of OsWRKY42 results in reduced expression of cell wall damage and salt stress induced jasmonic acid biosynthesis and response genes. OsWRKY42 expressing Arabidopsis lines exhibited enhanced tolerance to methyl jasmonate mediated growth inhibition.ConclusionThe results presented here suggest that OsWRKY42 regulates plant responses to either cell wall damage or salinity stress by acting as a negative regulator of jasmonic acid mediated responses.