P3N-PIPO, a Frameshift Product from the P3 Gene, Pleiotropically Determines the Virulence of Clover Yellow Vein Virus in both Resistant and Susceptible Peas.

UNLABELLED: Peas carrying the cyv1 recessive resistance gene are resistant to clover yellow vein virus (ClYVV) isolates No.30 (Cl-No.30) and 90-1 (Cl-90-1) but can be infected by a derivative of Cl-90-1 (Cl-90-1 Br2). The main determinant for the breaking of cyv1 resistance by Cl-90-1 Br2 is P3N-PIPO produced from the P3 gene via transcriptional slippage, and the higher level of P3N-PIPO produced by Cl-90-1 Br2 than by Cl-No.30 contributes to the breaking of resistance. Here we show that P3N-PIPO is also a major virulence determinant in susceptible peas that possess another resistance gene, Cyn1, which does not inhibit systemic infection with ClYVV but causes hypersensitive reaction-like lethal systemic cell death. We previously assumed that the susceptible pea cultivar PI 226564 has a weak allele of Cyn1 Cl-No.30 did not induce cell death, but Cl-90-1 Br2 killed the plants. Our results suggest that P3N-PIPO is recognized by Cyn1 and induces cell death. Unexpectedly, heterologously strongly expressed P3N-PIPO of Cl-No.30 appears to be recognized by Cyn1 in PI 226564. The level of P3N-PIPO accumulation from the P3 gene of Cl-No.30 was significantly lower than that of Cl-90-1 Br2 in a Nicotiana benthamiana transient assay. Therefore, Cyn1-mediated cell death also appears to be determined by the level of P3N-PIPO. The more efficiently a ClYVV isolate broke cyv1 resistance, the more it induced cell death systemically (resulting in a loss of the environment for virus accumulation) in susceptible peas carrying Cyn1, suggesting that antagonistic pleiotropy of P3N-PIPO controls the resistance breaking of ClYVV. IMPORTANCE: Control of plant viral disease has relied on the use of resistant cultivars; however, emerging mutant viruses have broken many types of resistance. Recently, we revealed that Cl-90-1 Br2 breaks the recessive resistance conferred by cyv1, mainly by accumulating a higher level of P3N-PIPO than that of the nonbreaking isolate Cl-No.30. Here we show that a susceptible pea line recognized the increased amount of P3N-PIPO produced by Cl-90-1 Br2 and activated the salicylic acid-mediated defense pathway, inducing lethal systemic cell death. We found a gradation of virulence among ClYVV isolates in a cyv1-carrying pea line and two susceptible pea lines. This study suggests a trade-off between breaking of recessive resistance (cyv1) and host viability; the latter is presumably regulated by the dominant Cyn1 gene, which may impose evolutionary constraints upon P3N-PIPO for overcoming resistance. We propose a working model of the host strategy to sustain the durability of resistance and control fast-evolving viruses.

P3N-PIPO of Clover yellow vein virus exacerbates symptoms in pea infected with white clover mosaic virus and is implicated in viral synergism.

Mixed infection of pea (Pisum sativum) with Clover yellow vein virus (ClYVV) and White clover mosaic virus (WClMV) led to more severe disease symptoms (a phenomenon called viral synergism). Similar to the mixed ClYVV/WClMV infection, a WClMV-based vector encoding P3N-PIPO of ClYVV exacerbated the disease symptoms. Infection with the WClMV vector encoding ClYVV HC-Pro (a suppressor of RNA silencing involved in potyviral synergisms), also resulted in more severe symptoms, although to a lesser extent than infection with the vector encoding P3N-PIPO. Viral genomic RNA accumulated soon after inoculation (at 2 and 4 days) at higher levels in leaves inoculated with WClMV encoding HC-Pro but at lower levels in leaves inoculated with WClMV encoding P3N-PIPO than in peas infected with WClMV encoding GFP. Our results suggest that ClYVV P3N-PIPO is involved in the synergism between ClYVV and WClMV during pea infection through an unknown mechanism different from suppression of RNA silencing.

Quantitative and qualitative involvement of P3N-PIPO in overcoming recessive resistance against Clover yellow vein virus in pea carrying the cyv1 gene.

In pea carrying cyv1, a recessive gene for resistance to Clover yellow vein virus (ClYVV), ClYVV isolate Cl-no30 was restricted to the initially infected cells, whereas isolate 90-1 Br2 overcame this resistance. We mapped the region responsible for breaking of cyv1-mediated resistance by examining infection of cyv1 pea with chimeric viruses constructed from parts of Cl-no30 and 90-1 Br2. The breaking of resistance was attributed to the P3 cistron, which is known to produce two proteins: P3, from the main open reading frame (ORF), and P3N-PIPO, which has the N-terminal part of P3 fused to amino acids encoded by a small open reading frame (ORF) called PIPO in the +2 reading frame. We introduced point mutations that were synonymous with respect to the P3 protein but nonsynonymous with respect to the P3N-PIPO protein, and vice versa, into the chimeric viruses. Infection of plants with these mutant viruses revealed that both P3 and P3N-PIPO were involved in overcoming cyv1-mediated resistance. Moreover, P3N-PIPO quantitatively affected the virulence of Cl-no30 in cyv1 pea. Additional expression in trans of the P3N-PIPO derived from Cl-no30, using White clover mosaic virus as a vector, enabled Cl-no30 to move to systemic leaves in cyv1 pea. Susceptible pea plants infected with chimeric ClYVV possessing the P3 cistron of 90-1 Br2, and which were therefore virulent toward cyv1 pea, accumulated more P3N-PIPO than did those infected with Cl-no30, suggesting that the higher level of P3N-PIPO in infected cells contributed to the breaking of resistance by 90-1 Br2. This is the first report showing that P3N-PIPO is a virulence determinant in plants resistant to a potyvirus.