Cowpea Veinal Necrosis Disease: Identification of the Associated Orthotospoviral Strain and Subcellular Localization of Orthotospoviral N and NSs Proteins

The increasing occurrence of virus disease in cowpea raises concerns about the emergence of new or expanding viral threats in the production of vegetable and pulse crops. During the summer of 2022, plants showing veinal necrosis disease on cowpea were observed at the experimental farm of the Indian Agricultural Research Institute (IARI), New Delhi. To elucidate the natural occurrence of groundnut bud necrosis virus (GBNV) associated with vein necrosis disease in cowpea, this study employed biological and molecular approaches, including the characterization and in silico prediction of structural and functional features of the N and NSs proteins, as well as subcellular localization studies in both natural and experimental host systems. Mechanical transmission successfully reproduced the typical disease as observed in the field on cowpea plants (cv. Pusa Komal). Molecular diagnosis using RT-PCR indicated the presence of GBNV in the diseased plant samples. Cloning and sequencing of the near-complete small RNA segment of GBNV revealed 98.9% nucleotide and > 99% amino acid identity in the nucleocapsid (N) gene, and 98% nucleotide and 99% amino acid identity for the non-structural silencing suppressor (NSs) gene, compared to known GBNV isolates. Phylogenetic analysis further placed the associated virus strain within the major clade of GBNV in plants. Further, the N and NSs proteins were successfully transiently expressed in plant and found localized in the cytoplasm of cowpea and Nicotiana benthamiana, the natural and experimental host of the virus respectively. The experimental reproduction of the disease and characterization of the N and NSs genes of the virus associated with pathogenesis revealed the association of the GBNV strain in the occurrence of veinal necrosis of cowpea. For the first time, functional motifs within the N and NSs genes were comprehensively predicted through bioinformatic analyses and their subcellular localization was examined in both natural and experimental hosts using confocal microscopy.