Overexpression of VpEIFP1, a novel F-box/Kelch-repeat protein from wild Chinese Vitis pseudoreticulata, confers higher tolerance to powdery mildew by inducing thioredoxin z proteolysis.

An F-box protein (VpEIFP1) induced by Erysiphe necator was isolated from Vitis pseudoreticulata, a wild Chinese grapevine species naturally resistant to powdery mildew (PM). It contains an F-box domain and two Kelch-repeat motifs. Expression profiles indicate the VpEIFP1 is strongly induced at both transcriptional and translational levels by PM infection. A subcellular localisation assay showed that VpEIFP1 is predominantly located in the nucleus and cytoplasm. Overexpression of VpEIFP1 accelerated the accumulation of hydrogen peroxide (H2O2) and up-regulated the expressions of ICS2, NPR1 and PR1 involved in defence responses, resulting in suppression of PM germination and growth. As an F-box protein, VpEIFP1 interacts with thioredoxin z (VpTrxz) in the yeast-two-hybrid (Y2H) assay and in the bimolecular fluorescence complementation (BiFC) assay. Decreased amounts of VpTrxz protein in transgenic grapevine leaves overexpressing VpEIFP1 were restored by proteasome inhibitor MG132, implying that VpEIFP1 mediated VpTrxz for degradation through the SCFVpEIFP1 (Skp1-Cullin-F-box) E3 ubiquitin ligase complex. The RNA interference line of VpTrxz showed increased H2O2 accumulation following PM inoculation. We propose VpEIFP1 positively modulates the grapevine defence response to PM by inducing the degradation of VpTrxz via the ubiquitin/26S proteasome system.

VpPUB24, a novel gene from Chinese grapevine, Vitis pseudoreticulata, targets VpICE1 to enhance cold tolerance.

The ubiquitination system plays important roles in the degradation and modification of substrate proteins. In this study, we characterize a putative U-box type E3 ubiquitin ligase gene, VpPUB24 (plant U-box protein 24), from Chinese wild grapevine, Vitis pseudoreticulata accession Baihe-35-1. We show that VpPUB24 is induced by a number of stresses, especially cold treatment. Real-time PCR analysis indicated that the PUB24 transcripts were increased after cold stress in different grapevine species, although the relative expression level was different. In grapevine protoplasts, we found that VpPUB24 was expressed at a low level at 22 °C but accumulated rapidly following cold treatment. A yeast two-hybrid assay revealed that VpPUB24 interacted physically with VpICE1. Further experiments indicated that VpICE1 is targeted for degradation via the 26S proteasome and that the degradation is accelerated by VpHOS1, and not by VpPUB24. Immunoblot analyses indicated that VpPUB24 promotes the accumulation of VpICE1 and suppresses the expression of VpHOS1 to regulate the abundance of VpICE1. Furthermore, VpICE1 promotes transcription of VpPUB24 at low temperatures. We also found that VpPUB24 interacts with VpHOS1 in a yeast two-hybrid assay. Additionally, over-expression of VpPUB24 in Arabidopsis thaliana enhanced cold tolerance. Collectively, our results suggest that VpPUB24 interacts with VpICE1 to play a role in cold stress.

RING-H2-type E3 gene VpRH2 from Vitis pseudoreticulata improves resistance to powdery mildew by interacting with VpGRP2A.

Grapevine is one of the world's most important fruit crops. European cultivated grape species have the best fruit quality but show almost no resistance to powdery mildew (PM). PM caused by Uncinula necator is a harmful disease that has a significant impact on the economic value of the grape crop. In this study, we examined a RING-H2-type ubiquitin ligase gene VpRH2 that is associated with significant PM-resistance of Chinese wild-growing grape Vitis pseudoreticulata accession Baihe-35-1. The expression of VpRH2 was clearly induced by U. necator inoculation compared with its homologous gene VvRH2 in a PM-susceptible grapevine V. vinifera cv. Thompson Seedless. Using a yeast two-hybrid assay we confirmed that VpRH2 interacted with VpGRP2A, a glycine-rich RNA-binding protein. The degradation of VpGRP2A was inhibited by treatment with the proteasome inhibitor MG132 while VpRH2 did not promote the degradation of VpGRP2A. Instead, the transcripts of VpRH2 were increased by over-expressing VpGRP2A while VpRH2 suppressed the expression of VpGRP2A. Furthermore, VpGRP2A was down-regulated in both Baihe-35-1 and Thompson Seedless after U. necator inoculation. Specifically, we generated VpRH2 overexpression transgenic lines in Thompson Seedless and found that the transgenic plants showed enhanced resistance to powdery mildew compared with the wild-type. In summary, our results indicate that VpRH2 interacts with VpGRP2A and plays a positive role in resistance to powdery mildew.