Multiple downy mildew effectors target the stress-related NAC transcription factor LsNAC069 in lettuce.

To cause disease in lettuce, the biotrophic oomycete Bremia lactucae secretes potential RxLR effector proteins. Here we report the discovery of an effector-target hub consisting of four B. lactucae effectors and one lettuce protein target by a yeast-two-hybrid (Y2H) screening. Interaction of the lettuce tail-anchored NAC transcription factor, LsNAC069, with B. lactucae effectors does not require the N-terminal NAC domain but depends on the C-terminal region including the transmembrane domain. Furthermore, in Y2H experiments, B. lactucae effectors interact with Arabidopsis and potato tail-anchored NACs, suggesting that they are conserved effector targets. Transient expression of RxLR effector proteins BLR05 and BLR09 and their target LsNAC069 in planta revealed a predominant localization to the endoplasmic reticulum. Phytophthora capsici culture filtrate and polyethylene glycol treatment induced relocalization to the nucleus of a stabilized LsNAC069 protein, lacking the NAC-domain (LsNAC069ΔNAC ). Relocalization was significantly reduced in the presence of the Ser/Cys-protease inhibitor TPCK indicating proteolytic cleavage of LsNAC069 allows for relocalization. Co-expression of effectors with LsNAC069ΔNAC reduced its nuclear accumulation. Surprisingly, LsNAC069 silenced lettuce lines had decreased LsNAC069 transcript levels but did not show significantly altered susceptibility to B. lactucae. In contrast, LsNAC069 silencing increased resistance to Pseudomonas cichorii bacteria and reduced wilting effects under moderate drought stress, indicating a broad role of LsNAC069 in abiotic and biotic stress responses.

Induction of epigenetic variation in Arabidopsis by over-expression of DNA METHYLTRANSFERASE1 (MET1).

Epigenetic marks such as DNA methylation and histone modification can vary among plant accessions creating epi-alleles with different levels of expression competence. Mutations in epigenetic pathway functions are powerful tools to induce epigenetic variation. As an alternative approach, we investigated the potential of over-expressing an epigenetic function, using DNA METHYLTRANSFERASE1 (MET1) for proof-of-concept. In Arabidopsis thaliana, MET1 controls maintenance of cytosine methylation at symmetrical CG positions. At some loci, which contain dense DNA methylation in CG- and non-CG context, loss of MET1 causes joint loss of all cytosines methylation marks. We find that over-expression of both catalytically active and inactive versions of MET1 stochastically generates new epi-alleles at loci encoding transposable elements, non-coding RNAs and proteins, which results for most loci in an increase in expression. Individual transformants share some common phenotypes and genes with altered gene expression. Altered expression states can be transmitted to the next generation, which does not require the continuous presence of the MET1 transgene. Long-term stability and epigenetic features differ for individual loci. Our data show that over-expression of MET1, and potentially of other genes encoding epigenetic factors, offers an alternative strategy to identify epigenetic target genes and to create novel epi-alleles.

TET3-mediated demethylation in tomato activates expression of a CETS gene that stimulates vegetative growth.

Expression of the mammalian DNA demethylase enzyme TET3 in plants can be used to induce hypomethylation of DNA. In tomato lines that express a TET3 transgene, we observed distinct phenotypes including an increase in the length and number of leaves of primary shoots. As these changes resemble phenotypes observed in plants with strong expression of SELF PRUNING (SP), a member of the PEBP/CETS family, we investigated in TET3 lines the expression levels of members of the PEBP/CETS gene family, which affect shoot architecture and growth of sympodial units in tomato. We did not detect any changes in SP expression in TET3 lines, but for CEN1.1, a putative family member that has not been functionally characterized, we identified changes in gene expression that corresponded to hypomethylation in the upstream region. In tomato wild type, CEN1.1 is expressed in roots, petals, and shoot apices but not in mature leaves. In contrast, in TET3 transformants, the CEN1.1 gene became hypomethylated and activated in leaves. Ectopic expression of CEN1.1 in tomato caused similar phenotypes to those seen in TET3 transformants. Vegetative growth was increased, resulting both in a delay in inflorescence development and in an instability of the inflorescences, which frequently reverted to a vegetative state. Ectopic expression of CEN1.1 in Arabidopsis thaliana also caused floral repression. Our data suggest that the phenotypes observed in TET3 lines are a consequence of ectopic activation of CEN1.1, which promotes vegetative growth, and that CEN1.1 expression is sensitive to DNA methylation changes.