Anthocyanin production as a potential visual selection marker during plant transformation

A mutant allele of the transcription factor gene MYB10 from apple induces anthocyanin production throughout the plant. This gene, including its upstream promoter, gene coding region and terminator sequence, was introduced into apple, strawberry and potato plants to determine whether it could be used as a visible selectable marker for plant transformation as an alternative to chemically selectable markers, such as kanamycin resistance. After transformation, red coloured calli, red shoots and red well-growing plants were scored. Red and green shoots were harvested from apple explants and examined for the presence of the MYB10 gene by PCR analysis. Red shoots of apple explants always contained the MYB10 gene but not all MYB10 containing shoots were red. Strawberry plants transformed with the MYB10 gene showed anthocyanin accumulation in leaves and roots. No visible accumulation of anthocyanin could be observed in potato plants grown in vitro, even the ones carrying the MYB10 gene. However, acid methanol extracts of potato shoots or roots carrying the MYB10 gene contained up to four times higher anthocyanin content than control plants. Therefore anthocyanin production as result of the apple MYB10 gene can be used as a selectable marker for apple, strawberry and potato transformation, replacing kanamycin resistance

In the Solanaceae, a hierarchy of bHLHs confer distinct target specificity to the anthocyanin regulatory complex

The anthocyanin biosynthetic pathway is regulated by a transcription factor complex consisting of an R2R3 MYB, a bHLH, and a WD40. Although R2R3 MYBs belonging to the anthocyanin-activating class have been identified in many plants, and their role well elucidated, the subgroups of bHLH implicated in anthocyanin regulation seem to be more complex. It is not clear whether these potential bHLH partners are biologically interchangeable with redundant functions, or even if heterodimers are involved. In this study, AcMYB110, an R2R3 MYB isolated from kiwifruit (Actinidia sp.) showing a strong activation of the anthocyanin pathway in tobacco (Nicotiana tabacum) was used to examine the function of interacting endogenous bHLH partners. Constitutive expression of AcMYB110 in tobacco leaves revealed different roles for two bHLHs, NtAN1 and NtJAF13. A hierarchical mechanism is shown to control the regulation of transcription factors and consequently of the anthocyanin biosynthetic pathway. Here, a model is proposed for the regulation of the anthocyanin pathway in Solanaceous plants in which AN1 is directly involved in the activation of the biosynthetic genes, whereas JAF13 is involved in the regulation of AN1 transcription

Multiple copies of a simple MYB-binding site confers trans-regulation by specific flavonoid-related R2R3 MYBs in diverse species

In apple, the MYB transcription factor MYB10 controls the accumulation of anthocyanins. MYB10 is able to auto-activate its expression by binding its own promoter at a specific motif, the R1 motif. In some apple accessions a natural mutation, termed R6, has more copies of this motif within the MYB10 promoter resulting in stronger auto-activation and elevated anthocyanins. Here we show that other anthocyanin-related MYBs selected from apple, pear, strawberry, petunia, kiwifruit and Arabidopsis are able to activate promoters containing the R6 motif. To examine the specificity of this motif, members of the R2R3 MYB family were screened against a promoter harboring the R6 mutation. Only MYBs from subgroups 5 and 6 activate expression by binding the R6 motif, with these MYBs sharing conserved residues in their R2R3 DNA binding domains. Insertion of the apple R6 motif into orthologous promoters of MYB10 in pear (PcMYB10) and Arabidopsis (AtMY75) elevated anthocyanin levels. Introduction of the R6 motif into the promoter region of an anthocyanin biosynthetic enzyme F3′5′H of kiwifruit imparts regulation by MYB10. This results in elevated levels of delphinidin in both tobacco and kiwifruit. Finally, an R6 motif inserted into the promoter the vitamin C biosynthesis gene GDP-L-Gal phosphorylase increases vitamin C content in a MYB10-dependent manner. This motif therefore provides a tool to re-engineer novel MYB-regulated responses in plants

Multiple Repeats of a Promoter Segment Causes Transcription Factor Autoregulation in Red Apples[W]

Mutations in the genes encoding for either the biosynthetic or transcriptional regulation of the anthocyanin pathway have been linked to color phenotypes. Generally, this is a loss of function resulting in a reduction or a change in the distribution of anthocyanin. Here, we describe a rearrangement in the upstream regulatory region of the gene encoding an apple (Malus × domestica) anthocyanin-regulating transcription factor, MYB10. We show that this modification is responsible for increasing the level of anthocyanin throughout the plant to produce a striking phenotype that includes red foliage and red fruit flesh. This rearrangement is a series of multiple repeats, forming a minisatellite-like structure that comprises five direct tandem repeats of a 23-bp sequence. This MYB10 rearrangement is present in all the red foliage apple varieties and species tested but in none of the white fleshed varieties. Transient assays demonstrated that the 23-bp sequence motif is a target of the MYB10 protein itself, and the number of repeat units correlates with an increase in transactivation by MYB10 protein. We show that the repeat motif is capable of binding MYB10 protein in electrophoretic mobility shift assays. Taken together, these results indicate that an allelic rearrangement in the promoter of MYB10 has generated an autoregulatory locus, and this autoregulation is sufficient to account for the increase in MYB10 transcript levels and subsequent ectopic accumulation of anthocyanins throughout the plant

The eggplant AG91-25 recognizes the Type III-secreted effector RipAX2 to trigger resistance to bacterial wilt (Ralstonia solanacearum species complex).

International audienceTo deploy durable plant resistance, we must understand its underlying molecular mechanisms. Type III effectors (T3Es) and their recognition play a central role in the interaction between bacterial pathogens and crops. We demonstrate that the Ralstonia solanacearum species complex (RSSC) T3E ripAX2 triggers specific resistance in eggplant AG91-25, which carries the major resistance locus EBWR9. The eggplant accession AG91-25 is resistant to the wild-type R. pseudosolanacearum strain GMI1000, whereas a ripAX2 defective mutant of this strain can cause wilt. Notably, the addition of ripAX2 from GMI1000 to PSS4 suppresses wilt development, demonstrating that RipAX2 is an elicitor of AG91-25 resistance. RipAX2 has been shown previously to induce effector-triggered immunity (ETI) in the wild relative eggplant Solanum torvum, and its putative zinc (Zn)-binding motif (HELIH) is critical for ETI. We show that, in our model, the HELIH motif is not necessary for ETI on AG91-25 eggplant. The ripAX2 gene was present in 68.1% of 91 screened RSSC strains, but in only 31.1% of a 74-genome collection comprising R. solanacearum and R. syzygii strains. Overall, it is preferentially associated with R. pseudosolanacearum phylotype I. RipAX2(GMI1000) appears to be the dominant allele, prevalent in both R. pseudosolanacearum and R. solanacearum, suggesting that the deployment of AG91-25 resistance could control efficiently bacterial wilt in the Asian, African and American tropics. This study advances the understanding of the interaction between RipAX2 and the resistance genes at the EBWR9 locus, and paves the way for both functional genetics and evolutionary analyses