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      CRISPR/Cas9-mediated targeted mutagenesis of TAS4 and MYBA7 loci in grapevine rootstock 101-14

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          Abstract

          Pierce’s disease (PD) of grapevine ( Vitis vinifera) is caused by the bacterium Xylella fastidiosa and is vectored by xylem sap-sucking insects, whereas Grapevine Red Blotch Virus (GRBV) causes Red Blotch Disease and is transmitted in the laboratory by alfalfa leafhopper Spissistilus festinus. The significance of anthocyanin accumulations in distinct tissues of grapevine by these pathogens is unknown, but vector feeding preferences and olfactory cues from host anthocyanins may be important for these disease etiologies. Phosphate, sugar, and UV light are known to regulate anthocyanin accumulation via miR828 and Trans- Acting Small- interfering locus4 ( TAS4), specifically in grape by production of phased TAS4a/b/c small-interfering RNAs that are differentially expressed and target MYBA5/6/7 transcription factor transcripts for post-transcriptional slicing and antisense-mediated silencing. To generate materials that can critically test these genes’ functions in PD and GRBV disease symptoms, we produced transgenic grape plants targeting TAS4b and MYBA7 using CRISPR/Cas9 technology. We obtained five MYBA7 lines all with bi-allelic editing events and no off-targets detected at genomic loci with homology to the guide sequence. We obtained two independent edited TAS4b lines; one bi-allelic, the other heterozygous while both had fortuitous evidences of bi-allelic TAS4a off-target editing events at the paralogous locus. No visible anthocyanin accumulation phenotypes were observed in regenerated plants, possibly due to the presence of genetically redundant TAS4c and MYBA5/6 loci or absence of inductive environmental stress conditions. The editing events encompass single base insertions and di/trinucleotide deletions of Vvi- TAS4a/b and Vvi- MYBA7 at expected positions 3 nt upstream from the guideRNA proximal adjacent motifs NGG. We also identified evidences of homologous recombinations of TAS4a with TAS4b at the TAS4a off-target in one of the TAS4b lines, resulting in a chimeric locus with a bi-allelic polymorphism, supporting independent recombination events in transgenic plants associated with apparent high Cas9 activities. The lack of obvious visible pigment phenotypes in edited plants precluded pathogen challenge tests of the role of anthocyanins in host PD and GRBV resistance/tolerance mechanisms. Nonetheless, we demonstrate successful genome-editing of non-coding RNA and MYB transcription factor loci which can serve future characterizations of the functions of TAS4a/b/c and MYBA7 in developmental, physiological, and environmental biotic/abiotic stress response pathways important for value-added nutraceutical synthesis and pathogen responses of winegrape.

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          The online version of this article (10.1007/s11248-020-00196-w) contains supplementary material, which is available to authorized users.

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          Most cited references55

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          A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana.

          To better understand the diversity of small silencing RNAs expressed in plants, we employed high-throughput pyrosequencing to obtain 887,000 reads corresponding to Arabidopsis thaliana small RNAs. They represented 340,000 unique sequences, a substantially greater diversity than previously obtained in any species. Most of the small RNAs had the properties of heterochromatic small interfering RNAs (siRNAs) associated with DNA silencing in that they were preferentially 24 nucleotides long and mapped to intergenic regions. Their density was greatest in the proximal and distal pericentromeric regions, with only a slightly preferential propensity to match repetitive elements. Also present were 38 newly identified microRNAs (miRNAs) and dozens of other plausible candidates. One miRNA mapped within an intron of DICER-LIKE 1 (DCL1), suggesting a second homeostatic autoregulatory mechanism for DCL1 expression; another defined the phase for siRNAs deriving from a newly identified trans-acting siRNA gene (TAS4); and two depended on DCL4 rather than DCL1 for their accumulation, indicating a second pathway for miRNA biogenesis in plants. More generally, our results revealed the existence of a layer of miRNA-based control beyond that found previously that is evolutionarily much more fluid, employing many newly emergent and diverse miRNAs, each expressed in specialized tissues or at low levels under standard growth conditions.
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            Uncovering small RNA-mediated responses to phosphate deficiency in Arabidopsis by deep sequencing.

            Recent studies have demonstrated the important role of plant microRNAs (miRNAs) under nutrient deficiencies. In this study, deep sequencing of Arabidopsis (Arabidopsis thaliana) small RNAs was conducted to reveal miRNAs and other small RNAs that were differentially expressed in response to phosphate (Pi) deficiency. About 3.5 million sequence reads corresponding to 0.6 to 1.2 million unique sequence tags from each Pi-sufficient or Pi-deficient root or shoot sample were mapped to the Arabidopsis genome. We showed that upon Pi deprivation, the expression of miR156, miR399, miR778, miR827, and miR2111 was induced, whereas the expression of miR169, miR395, and miR398 was repressed. We found cross talk coordinated by these miRNAs under different nutrient deficiencies. In addition to miRNAs, we identified one Pi starvation-induced DICER-LIKE1-dependent small RNA derived from the long terminal repeat of a retrotransposon and a group of 19-nucleotide small RNAs corresponding to the 5' end of tRNA and expressed at a high level in Pi-starved roots. Importantly, we observed an increased abundance of TAS4-derived trans-acting small interfering RNAs (ta-siRNAs) in Pi-deficient shoots and uncovered an autoregulatory mechanism of PAP1/MYB75 via miR828 and TAS4-siR81(-) that regulates the biosynthesis of anthocyanin. This finding sheds light on the regulatory network between miRNA/ta-siRNA and its target gene. Of note, a substantial amount of miR399* accumulated under Pi deficiency. Like miR399, miR399* can move across the graft junction, implying a potential biological role for miR399*. This study represents a comprehensive expression profiling of Pi-responsive small RNAs and advances our understanding of the regulation of Pi homeostasis mediated by small RNAs.
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              NewAgrobacterium helper plasmids for gene transfer to plants

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                Author and article information

                Contributors
                chris.rock@ttu.edu
                Journal
                Transgenic Res
                Transgenic Res
                Transgenic Research
                Springer International Publishing (Cham )
                0962-8819
                1573-9368
                23 April 2020
                23 April 2020
                2020
                : 29
                : 3
                : 355-367
                Affiliations
                GRID grid.264784.b, ISNI 0000 0001 2186 7496, Department of Biological Sciences, , Texas Tech University, ; Lubbock, TX 79409-3131 USA
                Author information
                http://orcid.org/0000-0002-2090-0650
                Article
                196
                10.1007/s11248-020-00196-w
                7283210
                32328868
                50b54b7e-97eb-4406-a20e-1ef1ade4716d
                © The Author(s) 2020

                Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 19 June 2019
                : 21 February 2020
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100006759, California Department of Food and Agriculture;
                Award ID: 15-0214-SA
                Award ID: 17-0514-SA
                Award Recipient :
                Categories
                Brief Communication
                Custom metadata
                © Springer Nature Switzerland AG 2020

                Genetics
                myb transcription factor,microrna,flavonoids,genome editing,off-target editing,rna interference,genetically modified organisms

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