Genome-wide identification and characterization of Chitinase gene family in Brassica juncea and Camelina sativa in response to Alternaria brassicae.

Chitinases belong to the group of Pathogenesis-related (PR) proteins that provides protection against fungal pathogens. This study presents the, genome-wide identification and characterization of chitinase gene family in two important oilseed crops B. juncea and C. sativa belonging to family Brassicaceae. We have identified 47 and 79 chitinase genes in the genomes of B. juncea and C. sativa, respectively. Phylogenetic analysis of chitinases in both the species revealed four distinct sub-groups, representing different classes of chitinases (I-V). Microscopic and biochemical study reveals the role of reactive oxygen species (ROS) scavenging enzymes in disease resistance of B. juncea and C. sativa. Furthermore, qRT-PCR analysis showed that expression of chitinases in both B. juncea and C. sativa was significantly induced after Alternaria brassicae infection. However, the fold change in chitinase gene expression was considerably higher in C. sativa compared to B. juncea, which further proves their role in C. sativa disease resistance to A. brassicae. This study provides comprehensive analysis on chitinase gene family in B. juncea and C. sativa and in future may serve as a potential candidate for improving disease resistance in B. juncea through transgenic approach.

Isolation and characterization of systemic acquired resistance marker gene PR1 and its promoter from Brassica juncea.

Systemic acquired resistance (SAR) is an inducible defense response in plants that provides enhanced resistance against a variety of pathogens. In this regard, SAR marker gene PR1 (pathogenesis-related gene 1) was isolated from Brassica juncea and was named as BjPR1. The amino acid sequence of BjPR1 protein showed 99, 92, and 78% similarity with known PR1 proteins of Brassica rapa, Brassica napus, and Arabidopsis thaliana, respectively. Quantitative real-time PCR (qRT-PCR) analysis showed increased expression of BjPR1 gene both in local (infected) and distal (non-infected) leaves of B. juncea after Alternaria brassicae infection, whereas mechanical wounding showed expression only in local (wounded) leaves but not in distal (unwounded) leaves. Moreover, BjPR1 gene was strongly induced by salicylic acid (SA), whereas no such induction was observed following jasmonic acid (JA) or abscisic acid (ABA) treatments. To further elucidate gene regulation pattern of BjPR1, 2 kb promoter region of BjPR1 was isolated and subjected to in silico analysis which identified many potential cis-regulatory elements associated with plant defense as well as signaling pathways. The transient GUS expression analysis showed strong expression of GUS gene driven by BjPR1 promoter after SA treatment, while as ABA and JA downregulates GUS gene expression compared to control. In addition, BjPR1 promoter was significantly induced by wounding at local tissues. Hence, these results highlight the multiple role of BjPR1 gene in response to biotic and abiotic stresses. In addition, the present study also reported BjPR1 promoter as stress-specific inducible promoter that can be ideal candidate for controlling the expression of biotic stress response genes in transgenic plants.

Pathogenesis-related proteins and peptides as promising tools for engineering plants with multiple stress tolerance.

Pathogenesis-related (PR) proteins and antimicrobial peptides (AMPs) are a group of diverse molecules that are induced by phytopathogens as well as defense related signaling molecules. They are the key components of plant innate immune system especially systemic acquired resistance (SAR), and are widely used as diagnostic molecular markers of defense signaling pathways. Although, PR proteins and peptides have been isolated much before but their biological function remains largely enigmatic despite the availability of new scientific tools. The earlier studies have demonstrated that PR genes provide enhanced resistance against both biotic and abiotic stresses, which make them one of the most promising candidates for developing multiple stress tolerant crop varieties. In this regard, plant genetic engineering technology is widely accepted as one of the most fascinating approach to develop the disease resistant transgenic crops using different antimicrobial genes like PR genes. Overexpression of PR genes (chitinase, glucanase, thaumatin, defensin and thionin) individually or in combination have greatly uplifted the level of defense response in plants against a wide range of pathogens. However, the detailed knowledge of signaling pathways that regulates the expression of these versatile proteins is critical for improving crop plants to multiple stresses, which is the future theme of plant stress biology. Hence, this review provides an overall overview on the PR proteins like their classification, role in multiple stresses (biotic and abiotic) as well as in various plant defense signaling cascades. We also highlight the success and snags of transgenic plants expressing PR proteins and peptides.

Expression analysis of chitinase upon challenge inoculation to Alternaria wounding and defense inducers in Brassica juncea.

Chitinases are the hydrolytic enzymes which belong to the pathogenesis-related (PR) protein family and play an important role not only in plant defense but also in various abiotic stresses. However, only a limited number of chitinase genes have been characterised in B. juncea. In this study, we have characterised B. juncea class IV chitinase gene (accession no EF586206) in response to fungal infection, salicylic acid (SA), jasmonic acid (JA) treatments and wounding. Gene expression studies revealed that the transcript levels of Bjchitinase (BjChp) gene increases significantly both in local and distal tissues after Alternaria infection. Bjchitinase gene was also induced by jasmonic acid and wounding but moderately by salicylic acid. A 2.5 kb class IV chitinase promoter of this gene was isolated from B. juncea by Genome walking (accession no KF055403.1). In-silico analysis of this promoter revealed a number of conserved cis-regulatory elements related to defense, wounding and signalling molecules like SA, and JA. For validation, chitinase promoter was fused to the GUS gene, and the resultant construct was then introduced into Arabidopsis plants. Histochemical analysis of T2 transgenic Arabidopsis plants showed that higher GUS activity in leaves after fungal infection, wounding and JA treatment but weakly by SA. GUS activity was seen in meristematic tissues, young leaves, seeds and siliques. Finally investigation has led to the identification of a pathogen-inducible, developmentally regulated and organ-specific promoter. Present study revealed that Bjchitinase (BjChp) promoter is induced during biotic and environmental stress and it can be used in developing finely tuned transgenics.

Overexpression of NPR1 in Brassica juncea Confers Broad Spectrum Resistance to Fungal Pathogens.

Brassica juncea (Indian mustard) is a commercially important oil seed crop, which is highly affected by many biotic stresses. Among them, Alternaria leaf blight and powdery mildew are the most devastating diseases leading to huge yield losses in B. juncea around the world. In this regard, genetic engineering is a promising tool that may possibly allow us to enhance the B. juncea disease resistance against these pathogens. NPR1 (non-expressor of pathogen-related gene 1) is a bonafide receptor of salicylic acid (SA) which modulates multiple immune responses in plants especially activation of induced and systemic acquired resistance (SAR). Here, we report the isolation and characterization of new NPR1 homolog (BjNPR1) from B. juncea. The phylogenetic tree constructed based on the deduced sequence of BjNPR1 with homologs from other species revealed that BjNPR1 grouped together with other known NPR1 proteins of Cruciferae family, and was nearest to B. napus. Furthermore, expression analysis showed that BjNPR1 was upregulated after SA treatment and fungal infection but not by jasmonic acid or abscisic acid. To understand the defensive role of this gene, we generated B. juncea transgenic lines overexpressing BjNPR1, and further confirmed by PCR and Southern blotting. The transgenic lines showed no phenotypic abnormalities, and constitutive expression of BjNPR1 activates defense signaling pathways by priming the expression of antifungal PR genes. Moreover, BjNPR1 transgenic lines showed enhanced resistance to Alternaria brassicae and Erysiphe cruciferarum as there was delay in symptoms and reduced disease severity than non-transgenic plants. In addition, the rate of disease spreading to uninfected or distal parts was also delayed in transgenic plants thus suggesting the activation of SAR. Altogether, the present study suggests that BjNPR1 is involved in broad spectrum of disease resistance against fungal pathogens.