Shoot transcriptome revealed widespread differential expression and potential molecular mechanisms of chickpea (Cicer arietinum L.) against Fusarium wilt.

Introduction: The yield of chickpea is severely hampered by infection wilt caused by several races of Fusarium oxysporum f. sp. ciceris (Foc). Methods: To understand the underlying molecular mechanisms of resistance against Foc4 Fusarium wilt, RNA sequencing-based shoot transcriptome data of two contrasting chickpea genotypes, namely KWR 108 (resistant) and GL 13001 (susceptible), were generated and analyzed. Results and Discussion: The shoot transcriptome data showed 1,103 and 1,221 significant DEGs in chickpea genotypes KWR 108 and GL 13001, respectively. Among these, 495 and 608 genes were significantly down and up-regulated in genotypes KWR 108, and 427 and 794 genes were significantly down and up-regulated in genotype GL 13001. The gene ontology (GO) analysis of significant DEGs was performed and the GO of the top 50 DEGs in two contrasting chickpea genotypes showed the highest cellular components as membrane and nucleus, and molecular functions including nucleotide binding, metal ion binding, transferase, kinase, and oxidoreductase activity involved in biological processes such as phosphorylation, oxidation-reduction, cell redox homeostasis process, and DNA repair. Compared to the susceptible genotype which showed significant up-regulation of genes involved in processes like DNA repair, the significantly up-regulated DEGs of the resistant genotypes were involved in processes like energy metabolism and environmental adaptation, particularly host-pathogen interaction. This indicates an efficient utilization of environmental adaptation pathways, energy homeostasis, and stable DNA molecules as the strategy to cope with Fusarium wilt infection in chickpea. The findings of the study will be useful in targeting the genes in designing gene-based markers for association mapping with the traits of interest in chickpea under Fusarium wilt which could be efficiently utilized in marker-assisted breeding of chickpea, particularly against Foc4 Fusarium wilt.

CRISPR/Cas9 mediated genome editing tools and their possible role in disease resistance mechanism.

Several phytopathogens have detrimental effects on crop production and productivity potentially threatening global food security. Studying the genetic mechanisms of virulence in phytopathogens is vital to assist in their management. Genome editing tools are paving their fascinating roles from the first-generation site-specific nucleases ZNF and TALEN to the current generation clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein9. The discovery of CRISPR/Cas9 has revolutionised the understanding of resistance as well as the susceptibility mechanism against phytopathogens in crop plants. This emerging tool allows researchers to perform precise genome manipulation, genetic screening, regulation, and correction to develop resistance in crop plants with fewer off-target effects. It provides a new opportunity for disease improvement and strengthens the resistant breeding programme. CRISPR/Cas9-based targeted gene manipulation and its enormous application potential as well as the challenges for developing transgene-free disease-resistant crop plants have been discussed in this review.

Heterologous expression and characterization of recombinant OsCDR1, a rice aspartic proteinase involved in disease resistance.

The Oryza sativa constitutive disease resistance 1 (OsCDR1) gene product is an aspartic proteinase that has been implicated in disease resistance signaling. This apoplastic enzyme is a member of the group of 'atypical' plant aspartic proteinases. Recombinant OsCDR1 expressed in Escherichia coli exhibited protease activity against succinylated-casein substrate. Inactivating the enzyme through modification of an aspartate residue present in the deduced active site completely abolished its proteinase activity. Infiltration of the OsCDR1 fusion protein into leaves of Arabidopsis plants induced PR2 transcripts in both the infiltrated leaf (primary) and in non-treated secondary leaves while the inactive recombinant protein failed to induce either local or systemic PR2. These findings demonstrate that OsCDR1 is capable of inducing systemic defense responses in plants.

Overexpression of rice (Oryza sativa L.) OsCDR1 leads to constitutive activation of defense responses in rice and Arabidopsis.

Plant aspartic proteases (AP) play key roles in the regulation of biological processes, such as the recognition of pathogens and pests and the induction of effective defense responses. A large number of AP (>400) have been identified in silico in the rice genome. None have previously been isolated and functionally characterized for their involvement in disease resistance. We describe here the isolation and characterization of a gene (OsCDR1) from rice which encodes a predicted aspartate protease. Expression of OsCDR1 was activated upon treatments with benzothiadiazole and salicylic acid, which are signal molecules in plant disease resistance responses. Ectopic expression of OsCDR1 in Arabidopsis and rice conferred enhanced resistance against bacterial and fungal pathogens. The enhanced disease resistance observed in transgenic plants was correlated with induction of pathogenesis-related gene expression and was shown by mutational analysis to be dependent on AP activity of the transgene-encoded product. OsCDR1 accumulates in intercellular fluids (IF) in transgenic plants. Infiltration of IF from transgenic Arabidopsis plants into leaves of wild-type (WT) Arabidopsis induced the systemic defense response. These results demonstrate the conservation of CDR1 function between rice and Arabidopsis during the disease resistance response.

Expression of Dm-AMP1 in rice confers resistance to Magnaporthe oryzae and Rhizoctonia solani.

Magnaporthe oryzae and Rhizoctonia solani, are among the most important pathogens of rice, severely limiting its productivity. Dm-AMP1, an antifungal plant defensin from Dahlia merckii, was expressed in rice (Oryza sativa L. sp. indica cv. Pusa basmati 1) using Agrobacterium tumefaciens-mediated transformation. Expression levels of Dm-AMP1 ranged from 0.43% to 0.57% of total soluble protein in transgenic plants. It was observed that constitutive expression of Dm-AMP1 suppresses the growth of M. oryzae and R. solani by 84% and 72%, respectively. Transgenic expression of Dm-AMP1 was not accompanied by an induction of pathogenesis-related (PR) gene expression, indicating that the expression of DmAMP1 directly inhibits the pathogen. The results of in vitro, in planta and microscopic analyses suggest that Dm-AMP1 expression has the potential to provide broad-spectrum disease resistance in rice.