Inhibition of multiple defense responsive pathways by CaWRKY70 transcription factor promotes susceptibility in chickpea under Fusarium oxysporum stress condition.

BACKGROUND: Suppression and activation of plant defense genes is comprehensively regulated by WRKY family transcription factors. Chickpea, the non-model crop legume suffers from wilt caused by Fusarium oxysporum f. sp. ciceri Race1 (Foc1), defense response mechanisms of which are poorly understood. Here, we attempted to show interaction between WRKY70 and several downstream signaling components involved in susceptibility/resistance response in chickpea upon challenge with Foc1. RESULTS: In the present study, we found Cicer arietinum L. WRKY70 (CaWRKY70) negatively governs multiple defense responsive pathways, including Systemic Acquired Resistance (SAR) activation in chickpea upon Foc1 infection. CaWRKY70 is found to be significantly accumulated at shoot tissues of susceptible (JG62) chickpea under Foc1 stress and salicylic acid (SA) application. CaWRKY70 overexpression promotes susceptibility in resistant chickpea (WR315) plants to Foc1 infection. Transgenic plants upon Foc1 inoculation demonstrated suppression of not only endogenous SA concentrations but expression of genes involved in SA signaling. CaWRKY70 overexpressing chickpea roots exhibited higher ion-leakage and Foc1 biomass accumulation compared to control transgenic (VC) plants. CaWRKY70 overexpression suppresses H2O2 production and resultant reactive oxygen species (ROS) induced cell death in Foc1 infected chickpea roots, stem and leaves. Being the nuclear targeted protein, CaWRKY70 suppresses CaMPK9-CaWRKY40 signaling in chickpea through its direct and indirect negative regulatory activities. Protein-protein interaction study revealed CaWRKY70 and CaRPP2-like CC-NB-ARC-LRR protein suppresses hyper-immune signaling in chickpea. Together, our study provides novel insights into mechanisms of suppression of the multiple defense signaling components in chickpea by CaWRKY70 under Foc1 stress. CONCLUSION: CaWRKY70 mediated defense suppression unveils networking between several immune signaling events negatively affecting downstream resistance mechanisms in chickpea under Foc1 stress.

CaMPK9 increases the stability of CaWRKY40 transcription factor which triggers defense response in chickpea upon Fusarium oxysporum f. sp. ciceri Race1 infection.

KEY MESSAGE: Physical interaction and phosphorylation by CaMPK9 protects the degradation of CaWRKY40 that induces resistance response in chickpea to Fusarium wilt disease by modulating the transcription of defense responsive genes. WRKY transcription factors (TFs) are the global regulators of plant defense signaling that modulate immune responses in host plants by regulating transcription of downstream target genes upon challenged by pathogens. However, very little is known about immune responsive role of Cicer arietinum L. (Ca) WRKY TFs particularly. Using two contrasting chickpea genotypes with respect to resistance against Fusarium oxysporum f. sp. ciceri Race1 (Foc1), we demonstrate transcript accumulation of different CaWRKYs under multiple stresses and establish that CaWRKY40 triggers defense. CaWRKY40 overexpressing chickpea mounts resistance to Foc1 by positively modulating the defense related gene expression. EMSA, ChIP assay and real-time PCR analyses suggest CaWRKY40 binds at the promoters and positively regulates transcription of CaDefensin and CaWRKY33. Further studies revealed that mitogen Activated Protein Kinase9 (CaMPK9) phosphorylates CaWRKY40 by directly interacting with its two canonical serine residues. Interestingly, CaMPK9 is unable to interact with CaWRKY40 when the relevant two serine residues were replaced by alanine. Overexpression of serine mutated WRKY40 isoform in chickpea fails to provide resistance against Foc1. Mutated WRKY40Ser.224/225 to AA overexpressing chickpea resumes its ability to confer resistance against Foc1 after application of 26S proteasomal inhibitor MG132, suggests that phosphorylation is essential to protect CaWRKY40 from proteasomal degradation. CaMPK9 silencing also led to susceptibility in chickpea to Foc1. Altogether, our results elucidate positive regulatory roles of CaMPK9 and CaWRKY40 in modulating defense response in chickpea upon Foc1 infection.

Chickpea WRKY70 Regulates the Expression of a Homeodomain-Leucine Zipper (HD-Zip) I Transcription Factor CaHDZ12, which Confers Abiotic Stress Tolerance in Transgenic Tobacco and Chickpea.

Drought and salinity are the two major environmental constraints that severely affect global agricultural productivity. Plant-specific HD-Zip transcription factors are involved in plant growth, development and stress responses. In the present study, we explored the functional characteristics and regulation of a novel HD-Zip (I) gene from chickpea, CaHDZ12, in response to water-deficit and salt-stress conditions. Transgenic tobacco lines over-expressing CaHDZ12 exhibited improved tolerance to osmotic stresses and increased sensitivity to abscisic acid (ABA). Physiological compatibility of transgenic lines was found to be more robust compared to the wild-type plants under drought and salinity stress. Additionally, expression of several stress-responsive genes was significantly induced in CaHDZ12 transgenic plants. On the other hand, silencing of CaHDZ12 in chickpea resulted in increased sensitivity to salt and drought stresses. Analysis of different promoter deletion mutants identified CaWRKY70 transcription factor as a transcriptional regulator of CaHDZ12 expression. In vivo and in vitro interaction studies detected an association between CaWRKY70 and CaHDZ12 promoter during stress responses. Epigenetic modifications underlying histone acetylation at the CaHDZ12 promoter region play a significant role in stress-induced activation of this gene. Collectively, our study describes a crucial and unique mechanistic link between two distinct transcription factors in regulating plant adaptive stress response.

Monitoring the efficacy of mutated Allium sativum leaf lectin in transgenic rice against Rhizoctonia solani.

BACKGROUND: Rice sheath blight, caused by Rhizoctonia solani is one of the most devastating diseases of rice. It is associated with significant reduction in rice productivity worldwide. A mutant variant of mannose binding Allium sativum leaf agglutinin (mASAL) was previously reported to exhibit strong antifungal activity against R. solani. In this study, the mASAL gene has been evaluated for its in planta antifungal activity in rice plants. RESULTS: mASAL was cloned into pCAMBIA1301 binary vector under the control of CaMV35S promoter. It was expressed in an elite indica rice cv. IR64 by employing Agrobacterium tumefaciens-mediated transformation. Molecular analyses of transgenic plants confirmed the presence and stable integration of mASAL gene. Immunohistofluorescence analysis of various tissue sections of plant parts clearly indicated the constitutive expression of mASAL. The segregation pattern of mASAL transgene was observed in T1 progenies in a 3:1 Mendelian ratio. The expression of mASAL was confirmed in T0 and T1 plants through western blot analysis followed by ELISA. In planta bioassay of transgenic lines against R. solani exhibited an average of 55 % reduction in sheath blight percentage disease index (PDI). CONCLUSIONS: The present study opens up the possibility of engineering rice plants with the antifungal gene mASAL, conferring resistance to sheath blight.

Epigenetic and transcriptional control of chickpea WRKY40 promoter activity under Fusarium stress and its heterologous expression in Arabidopsis leads to enhanced resistance against bacterial pathogen.

Promoters of many defense related genes are enriched with W-box elements serving as binding sites for plant specific WRKY transcription factors. In this study, expression of WRKY40 transcription factor was analyzed in two contrasting susceptible (JG62) and resistant (WR315) genotypes of chickpea infected with Foc1. The resistant plants showed up-regulation of WRKY40 under Fusarium stress, whereas in susceptible plants WRKY40 expression was absent. Additionally, global changes in the histone modification patterns were studied in above two chickpea genotypes by immunoblotting and real-time PCR analyses under control and Fusarium infected conditions. Notably, region specific Histone 3 lysine 9 acetylation, a positive marker of transcription gets enriched at WRKY40 promoter during resistant interaction with Foc1. H3K9 Ac is less enriched at WRKY40 promoter in Foc1 infected susceptible plants. WRKY40 promoter activity was induced by jasmonic acid and pathogen treatment, while salicylic acid failed to stimulate such activity. Moreover, WRKY40 was found to bind to its own promoter and auto-regulates its activity. The present study also showed that heterologous over-expression of chickpea WRKY40 triggers defense response in Arabidopsis against Pseudomonas syringae. Overall, we present epigenetic and transcriptional control of WRKY40 in chickpea under Fusarium stress and its immunomodulatory role is tested in Arabidopsis.