Inducible expression of truncated NAC62 provides tolerance against Alternaria brassicicola and imparts developmental changes in Indian mustard.

Indian mustard (Brassica juncea) faces significant yield loss due to the 'Black Spot Disease,' caused by a fungus Alternaria brassicicola. In plants, NAC transcription factors (NAC TFs) are known for their roles in development and stress tolerance. One such NAC TF, NAC 62, was induced during A. brassicicola challenge in Sinapis alba, a non-host resistant plant against this fungus. Sequence analyses of BjuNAC62 from B. juncea showed that it belonged to the membrane-bound class of transcription factors. Gene expression study revealed differential protein processing of NAC62 between B. juncea and S. alba on pathogen challenge. Furthermore, NAC62 processing to 25 kDa protein was found to be unique to the resistant plant during pathogenesis. Conditional expression of BjuNAC62ΔC, which lacks its transmembrane domain, in B. juncea showed improved tolerance to A. brassicicola. BjuNAC62ΔC processing to 25 kDa product was also observed in tolerant transgenic plants. Additionally, transgenic plants showed induced expression of genes associated with defense-related phytohormone signaling pathways on pathogen challenge. Again, altered phenotypes suggest a possible developmental effect of BjuNAC62∆C in transgenic plants. The overall results suggest that the processing of BjuNAC62 might be playing a crucial role in resistance response against Black Spot disease by modulating defense-associated genes.

Overexpression of LYK4, a lysin motif receptor with non-functional kinase domain, enhances tolerance to Alternaria brassicicola and increases trichome density in Brassica juncea.

Lysin motif receptor-like kinases (LYKs) are involved in the recognition of chitin and activation of plant immune response. In this study, we found LYK4 to be strongly induced in resistant Sinapis alba compared with susceptible Brassica juncea on challenge with Alternaria brassicicola. In silico analysis and in vitro kinase assay revealed that despite the presence of canonical protein kinase fold, B.juncea LYK4 (BjLYK4) lacks several key residues of a prototype protein kinase which renders it catalytically inactive. Transient expression analysis confirmed that fluorescently tagged BjLYK4 localizes specifically to the plasma membrane. Overexpression (OE) of BjLYK4 in B. juncea enhanced tolerance against A. brassicicola. Interestingly, the OE lines also exhibited a novel trichome dense phenotype and increased jasmonic acid (JA) responsiveness. We further showed that many chitin responsive WRKY transcription factors and JA biosynthetic genes were strongly induced in the OE lines on challenge with the pathogen. Moreover, several JA inducible trichome developmental genes constituting the WD-repeat/bHLH/MYB activator complex were also upregulated in the OE lines compared with vector control and RNA interference line. These results suggest that BjLYK4 plays an essential role in chitin-dependent activation of defense response and chitin independent trichome development likely by influencing the JA signaling pathway.

Enhancement of ABA Sensitivity Through Conditional Expression of the ARF10 Gene in Brassica juncea Reveals Fertile Plants with Tolerance Against Alternaria brassicicola.

Concomitant increase of auxin-responsive factors ARF16 and ARF17, along with enhanced expression of ARF10 in resistant Sinapis alba compared with that in susceptible Brassica juncea upon challenge with Alternaria brassicicola, revealed that abscisic acid (ABA)-auxin crosstalk is a critical factor for resistance response. Here, we induced the ABA response through conditional expression of ARF10 in B. juncea using the A. brassicicola-inducible GH3.3 promoter. Induced ABA sensitivity caused by conditional expression of ARF10 in transgenic B. juncea resulted in tolerance against A. brassicicola and led to enhanced expression of several ABA-responsive genes without affecting the auxin biosynthetic gene expression. Compared with ABI3 and ABI4, ABI5 showed maximum upregulation in the most tolerant transgenic lines upon pathogen challenge. Moreover, elevated expression of ARF10 by different means revealed a direct correlation between ARF10 expression and the induction of ABI5 protein in B. juncea. Through in vitro DNA-protein experiments and chromosome immunoprecipitation using the ARF10 antibody, we demonstrated that ARF10 interacts with the auxin-responsive elements of the ABI5 promoter. This suggests that ARF10 may function as a modulator of ABI5 to induce ABA sensitivity and mediate the resistance response against A. brassicicola.