Genome-based identification of Pvr4 conferring resistance against potyvirus in Capsicum annuum

Abstract

Pvr4 is a resistance gene showing broad-spectrum resistance against multiple potyviruses, including Pepper mottle virus (PepMoV), Pepper severe mosaic virus (PepSMV), and Potato virus Y (PVY). Capsicum annuum landrace CM334 is known to have Pvr4, but it has not been cloned and the mechanism of resistance is unknown. To identify the avirulence factor in potyviruses corresponding to Pvr4, a total of eleven viral cistrons of PepMoV were expressed into potyvirus-resistant (Pvr4) and -susceptible (pvr4) pepper plants. Hypersensitive response (HR) was observed only when a RNA-dependent RNA polymerase (NIb) of PepMoV was expressed in Pvr4-haboring pepper leaves in a genotype-specific manner. In addition, the over-expression of NIb proteins of other potyviruses including PepSMV, PVY also induced HR in Pvr4-harboring pepper plants. These results indicate that NIbs of PepMoV, PepSMV, and PVY may play important roles as avirulence factors for Pvr4 in pepper plants. To identify Pvr4 resistance gene against potyvirus in pepper, genome-based cloning with two populations including BC1F3 and F2 populations was performed. High-density molecular markers including 32 co-dominant markers were developed in the TG420 marker region using tomato and pepper genome. Three SNP markers showed a co-segregation with Pvr4 in two populations and Pvr4 is located within 350 kb region containing sixteen annotated genes. Among them, eight genes were coiled-coil (CC) nucleotide-binding site leucine-rich repeat (NB-LRR) and they were clustered in this region. Transient over-expression of the eight NB-LRR type genes and NIb of PepMoV in susceptible pepper (C. annuum Jupiter) and Nicotiana benthamiana leaves revealed that only one gene (CA10g21170) induced HR at 2 dpi. The relative amounts of PepMoV RNA transcripts and protein were significantly suppressed in the leaves that transiently over-expressed of CA10g21170 after 3 dpi. Furthermore, CA10g21170 showed resistance against other potyviruses including PepSMV and PVY. Consequently, these results prove that CA10g21170 is indeed Pvr4 leading to recognize NIb and suppress PepMoV, PepSMV and PVY replication. Pvr4 consisted of seven exons and encodes a CC-NB-LRR type protein with 1746 amino acids. Genomic region of Pvr4 including exon and intron is 13,870 bp. In planta assays using the TRV-based gene silencing revealed that silencing of HSP90, SGT1 and RAR1 in N. benthamiana suppressed HR induced by Pvr4 and NIb of PepMoV. To confirm the heterologous in planta expression of Pvr4 in other plant, Pvr4-harboring transgenic potato were generated. Inoculation of virus validated that the replication of PVY-O was significantly suppressed in the transgenic upper leaves. Taken together, the cloned potyvirus resistance gene, Pvr4 could provide information for the application of broad-spectrum potyvirus resistance in crop breeding, as well as for understanding potyvirus resistance mechanisms in plants.