Can-miRn37a mediated suppression of ethylene response factors enhances the resistance of chilli against anthracnose pathogen Colletotrichum truncatum L.

Pepper anthracnose, caused by Colletotrichum species complex is the most destructive disease of chilli (Capsicum annuum L.). miRNAs are key modulators of transcriptional and post- transcriptional expression of genes during defense responses. In the present study, we performed a comparative miRNA profiling of susceptible (Arka Lohit-AL) and resistant (Punjab Lal-PL) chilli cultivars to identify 35 differentially expressed miRNAs that could be classified as positive, negative or basal regulators of defense against C. truncatum, the most potent anthracnose pathogen. Interestingly, a novel microRNA can-miRn37a was significantly induced in PL but largely repressed in AL genotype post pathogen attack. Subsequent over-expression of can-miRn37a in AL showed enhanced resistance to anthracnose, as evidenced by decreased fungal growth and induced expression of defense-related genes. Consequently, the expression of its three target genes encoding the ethylene response factors (ERFs) was down-regulated in PL as well as in the over-expression lines of AL genotypes. The ability of these targets to be regulated by can-miRn37a was further confirmed by transient co-expression in Nicotiana benthamiana. Additionally, the virus-induced silencing of the three targets in the susceptible AL cultivar revealed their role in fungal colonization and induction of C. truncatum pathogenicity in chilli. Taken together, our study suggests that can-miRn37a provides a potential miRNA mediated approach of engineering anthracnose resistance in chilli by repressing ERFs and preventing fungal colonization.

Multiple garlic (Allium sativum L.) microRNAs regulate the immunity against the basal rot fungus Fusarium oxysporum f. sp. Cepae.

The basal plate rot fungus, Fusarium oxysporum f. sp. cepae (FOC), is the most devastating pathogen posing a serious threat to garlic (Allium sativum L.) production worldwide. MicroRNAs (miRNAs) are key modulators of gene expression related to development and defense responses in eukaryotes. However, the miRNA species associated with garlic immunity against FOC are yet to be explored. In the present study, a small RNA library developed from FOC infected resistant garlic line was sequenced to identify immune responsive miRNAs. Forty-five miRNAs representing 39 conserved and six novel sequences responsive to FOC were detected. qRT-PCR analyses further classified them into three classes based on their expression patterns in susceptible line CBT-As11 and in the resistant line CBT-As153. North-blot analyses of six selective miRNAs confirmed the qRT-PCR results. Expression studies on a selective set of target genes revealed a negative correlation with the complementary miRNAs. Furthermore, transgenic garlic plant overexpresing miR164a, miR168a and miR393 showed enhanced resistance to FOC, as revealed by decreased fungal growth and up-regulated expression of defense-responsive genes. These results indicate that multiple miRNAs are involved in garlic immunity against FOC and that the overexpression of miR164a, miR168a and miR393 can augment garlic resistance to Fusarium basal rot infection.

Identification of Novel Source of Resistance and Differential Response of Allium Genotypes to Purple Blotch Pathogen, Alternaria porri (Ellis) Ciferri.

Purple blotch, caused by Alternaria porri (Ellis) Cifferi, is a serious disease incurring heavy yield losses in the bulb and seed crop of onion and garlic worldwide. There is an immediate need for identification of effective resistance sources for use in host resistance breeding. A total of 43 Allium genotypes were screened for purple blotch resistance under field conditions. Allium cepa accession 'CBT-Ac77' and cultivar 'Arka Kalyan' were observed to be highly resistant. In vitro inoculation of a selected set of genotypes with A. porri, revealed that 7 days after inoculation was suitable to observe the disease severity. In vitro screening of 43 genotypes for resistance to A. porri revealed two resistant lines. An additional 14 genotypes showed consistent moderate resistance in the field as well as in vitro evaluations. Among the related Allium species, A. schoenoprasum and A. roylei showed the least disease index and can be used for interspecific hybridization with cultivated onion. Differential reaction analysis of three A. porri isolates (Apo-Chiplima, Apn-Nasik, Apg-Guntur) in 43 genotypes revealed significant variation among the evaluated Allium species (P = 0.001). All together, the present study suggest that, the newly identified resistance sources can be used as potential donors for ongoing purple blotch resistance breeding program in India.

Mining, characterization and validation of EST derived microsatellites from the transcriptome database of Allium sativum L.

Expressed Sequence Tags (ESTs) with comprehensive transcript information are valuable resources for development of molecular markers as they are derived from conserved genic regions. The present study highlights the mining of EST database to deduce the class I hyper variable SSRs in A. sativum. From 21694 garlic EST sequences, 642 non-redundant SSRs were identified with an average frequency of 1 per 14.9 kb of garlic transcriptome. The most abundant SSR motifs were the mononucleotides (32.86%) followed by trinucleotides (28.50%) and dinucleotides (13.39%). Among the individual SSRs, (A/T)n accounted for the highest number (137; 21.33%) followed by (G/C)n (74; 11.52%) and (AAG)n (63;9.81%). Primers designed from a robust set of 7 AsESTSSRs resulted in the amplification of 63 polymorphic alleles in 14 accessions of garlic. The resolving power of the markers varied from 4.286 (AsSSR7) to 18.143 (AsSSR13) while the average marker index (MI) was 5.087. These EST-SSRs markers for garlic could be useful for the improvement of garlic linkage map and could be used for evaluating genetic variation and comparative genomics studies in Allium species.