Development of Molecular Marker Linked with Cercospora Leaf Spot (CLS) Disease Resistance in Vigna radiata, Cloning, and Expression for Evaluating Antifungal Activity against Cercospora canescens

Abstract

We developed a molecular marker for MAS of mungbean resistant varieties against CLS from the consensus sequence (MB-CLsRG) of identified RGAs (MB-ClsRCaG1 and MB-ClsRCaG2). The MB-CLsRG sequence-specific primer pair was used to screen Cercospora leaf spot (CLS) resistant varieties of mungbean in genomic analysis that showed congruency with phenotypic screening. Validation of molecular marker linkage with CLS resistance was performed using rtPCR in transcriptomic analysis. The sequenced PCR products showed 100% homology with MB-CLsRG sequence and putative disease resistance proteins that confirmed the linkage of molecular marker with CLS resistance in mungbean. The antifungal potential of MB-CLsRG gene encoding protein was assessed. The MB-CLsRG gene sequence was cloned in the E. coli expression vector for recombinant protein production. The recombinant protein was then investigated for its in vitro antifungal potential against Cercospora canescens. The in vitro investigation showed strong antifungal activity of recombinant protein as it restricted the growth of fungal mycelial mass. The results validated the linkage of developed marker with CLS-resistant mungbean varieties; therefore, it can be used to screen resistant varieties from a large population in MAS. Moreover, the recombinant protein of the MB-CLsRG gene sequence revealed antifungal potential, which proved the gene sequence could be suitable to use in transgenic plants technology to develop fungal-resistant transgenic crops.

Progress in the Use of Combined Omics for Mungbean Breeding Improvement and Its Potential in Promoting Resistance against Cercospora Leaf Spot

“…Moreover, in recently conducted research by a team of experts, Babar et al [42], the antifungal potential of MB-CLsRG gene-encoding protein was assessed to screen CLS-resistant mungbean varieties in genomic analysis that showed strong agreement with phenotypic selection. According to these authors, strong antifungal activity of recombinant protein encoded by the MB-CLsRG gene was observed as the protein inhibiting the growth of the mycelial mass of Cercospora canescens.…”

“…The amino acid, serine, can be changed to theorine in the mungbean resistance gene to CLS disease by the functional SNPs known as Indel in exon 8 located in the gene [61]. Antimicrobial peptides and other compounds derived from plants can suppress pathogenicity by direct detoxification or through inhibition of the activity of virulence factors [42]. Proteomics can be described as the study of the interactions, function, composition, and structures of proteins and their cellular activities where the automated, high-throughput functional and structural proteomics technologies use advanced materials such as multidimensional nuclear magnetic resonance spectroscopy and X-ray crystallography [124].…”

“…They then prokaryotically expressed and purified the two VrPGIP proteins and demonstrated that VrPGIP1 and VrPGIP2 play a critical role in bruchid resistance, probably through inhibiting polygalacturonase enzyme activity. Moreover, Babar et al [42] tested the antifungal activity of the purified protein assessed through the modified radial diffusion method, and then the purified protein (~15.4 kDa) exhibited a strong inhibitory effect on the mycelial growth of Cercospora canescens.…”

“…Some of such investigations were carried out to evaluate the efficacies of fungicides in reducing disease incidence and/or severity and yield value. However, applications of modern high-throughput technologies and the genomic information for resistance breeding are inadequate in mungbean when compared to cereals and other legumes [41,42], which is a critical component to recommend for mungbean improvement. Furthermore, evaluations of fungal diseases, specifically mungbean's CLS triggered by Cercospora canescens, have not been consolidated through the utilisation of combined omics such as genomics, transcriptomics, proteomics, metabolomics, and other advanced technologies.…”