The Effect of Transcription Factor MYB14 on Defense Mechanisms in Vitis quinquangularis-Pingyi.

In the current study, we identified a transcription factor, MYB14, from Chinese wild grape, Vitis quinquangularis-Pingyi (V. quinquangularis-PY), which could enhance the main stilbene contents and expression of stilbene biosynthesis genes (StSy/RS) by overexpression of VqMYB14. The promoter of VqMYB14 (pVqMYB14) was shown to be induced as part of both basal immunity (also called pathogen-associated molecular pattern (PAMP)-triggered immunity, PTI) and effector-triggered immunity (ETI), triggered by the elicitors flg22 and harpin, respectively. This was demonstrated by expression of pVqMYB14 in Nicotiana benthamiana and Vitis. We identified sequence differences, notably an 11 bp segment in pVqMYB14 that is important for the PTI/ETI, and particularly for the harpin-induced ETI response. In addition, we showed that activation of the MYB14 promoter correlates with differences in the expression of MYB14 and stilbene pattern induced by flg22 and harpin. An experimental model of upstream signaling in V. quinquangularis-PY is presented, where early defense responses triggered by flg22 and harpin partially overlap, but where the timing and levels differ. This translates into a qualitative difference with respect to patterns of stilbene accumulation.

Transcriptional regulation of stilbene synthases in grapevine germplasm differentially susceptible to downy mildew.

BACKGROUND: To limit the impact of the downy mildew disease of grapevine and reduce the need to recur to chemical treatments, an effective strategy might be recovering adaptive resistance traits in both cultivated and wild V. vinifera germplasm. Considering that stilbenes represent the most important class of phytoalexins in the Vitaceae, the constitutive expression and transcriptional activation of all the functional members of the stilbene synthase gene family were analysed in a group of nine grapevine genotypes following artificial infection with the oomycete Plasmopara viticola, the causal agent of the disease. In addition, in the same genotypes we analyzed the expression of genes encoding for two transcription factors involved in the transcriptional regulation of the stilbene synthase genes, namely VvMYB14 and VvMYB15, and of genes encoding for chalcone synthases. RESULTS: Downy mildew incidence and severity ranged from nihil to high in the grapevine genotypes considered, being low to moderate in a subgroup of V. vinifera genotypes. The constitutive expression of the stilbene synthase genes as well as the extent of their transcriptional activation following P. viticola inoculation appeared to be inversely related to the proneness to develop disease symptoms upon infection. In a specular manner, following P. viticola inoculation all the chalcone synthase genes were up-regulated in the susceptible grapevine genotypes and down-regulated in the resistant ones. The infection brought by P. viticola appeared to elicit a co-ordinated and sequential transcriptional activation of distinct stilbene synthase genes subsets, each of which may be regulated by a distinct and specific MYB transcription factor. CONCLUSIONS: The present results suggest that the induction of stilbene biosynthesis may contribute to the basal immunity against the downy mildew of grapevine, thus representing an adaptive resistance trait to recover, in both cultivated and wild V. vinifera germplasm. During the early stages of P. viticola infection, an antagonistic interaction between flavonol and stilbene biosynthesis might occur, whose outcome might determine the subsequent extent of disease symptoms. Further studies are needed to decipher the possible regulatory mechanisms involved in the antagonistic crosstalk between these two metabolic pathways in resistant and susceptible genotypes in response to P. viticola.

A specific allele of MYB14 in grapevine correlates with high stilbene inducibility triggered by Al3+ and UV-C radiation.

KEY MESSAGE: The structural differences of MYB14 promoter in two grapevine genotypes affect the expression of MYB14 and stilbene synthesis in response to Al3+ and UV-C radiation. Grapevines provide an important fruit crop worldwide, but production is often limited by pathogen infection. Stilbenes, a class of secondary metabolite, represent phytoalexins that contribute to defence against pathogens in many plants, including grapevine. It is known that the transcription factors MYB14 and MYB15 are required for the activation of the promoters of resveratrol synthase to regulate stilbene biosynthesis. In the current study, we observed that stilbene levels were more highly induced by Al3+ and UV-C radiation treatments in the cultivar Vitis labrusca 'Concord' than in the cultivar V. vinifera 'Cabernet Sauvignon'. We investigated whether genetic/structural variations in the MYB14 and MYB15 promoters between these two representative genotypes are responsible for the differences in stilbene accumulation. Significant differences in the structure and activity of the promoter of MYB14, but not MYB15 were identified between the two genotypes, following heterologous expression in Nicotiana benthamiana system and treatments with Al3+ and UV-C. Hydrogen peroxide (H2O2) was detected in Concord soon after the stress treatments, but after diphenyleneiodonium chloride pre-treatment, the expressing level of VlMYB14, the promoter activity of VlMYB14 and the accumulation of stilbenes was significantly reduced. A model is presented where the induction of MYB14 contributes to stilbene accumulation in Concord following Al3+ and UV-C treatments involving reactive oxygen species (ROS) production as an early signal.

An ancestral allele of grapevine transcription factor MYB14 promotes plant defence.

Stilbene synthase is a key enzyme for the production of the phytoalexin resveratrol. Some clones of Vitis sylvestris, a wild European grapevine species which is almost extinct, have been shown to accumulate more resveratrol in response to different forms of stress. In the current study, we asked whether the induction of stilbene synthase transcripts in Hoe29, one of the V. sylvestris clones with elevated stilbene inducibility, might result from the elevated induction of the transcription factor MYB14. The MYB14 promoter of Hoe29 and of Ke83 (a second stilbene-inducible genotype) harboured distinct regions and were applied to a promoter-reporter system. We show that stilbene synthase inducibility correlates with differences in the induction of MYB14 transcripts for these two genotypes. Both alleles were induced by UV in a promoter-reporter assay, but only the MYB14 promoter from Hoe29 was induced by flg22, consistent with the stilbene synthase expression of the donor genotypes, where both respond to UV but only Hoe29 is responsive to Plasmopara viticola during defence. We mapped upstream signals and found that a RboH-dependent oxidative burst, calcium influx, a MAPK cascade, and jasmonate activated the MYB14 promoter, whereas salicylic acid was ineffective. Our data suggest that the Hoe29 allele of the MYB14 promoter has potential as a candidate target for resistance breeding.

A gain-of-function T-DNA insertion mutant of Marchantia polymorpha hyper-accumulates flavonoid riccionidin A.

Marchantia polymorpha is a model liverwort for which many molecular biological techniques are now available. We previously developed the S-AgarTrap method for easy genetic transformation of M. polymorpha using spores. In this study, we report production of a T-DNA insertion mutant library (approx. 10,000 lines) for M. polymorpha using the S-AgarTrap method. We further isolate and characterize a gain-of-function mutant that hyper-accumulates the flavonoid riccionidin A. The present study demonstrates that the S-AgarTrap-mediated production of a T-DNA insertion mutant library is a powerful tool for molecular biology in M. polymorpha.