Factors influencing Barley Stripe Mosaic Virus-mediated gene silencing in wheat

Virus induced gene silencing (VIGS) is a technology that has been used primarily to target the virus genome in infected plants. However, if the virus genome carries inserts derived from the host plant, the system could be employed to target the mRNAs corresponding to the host gene. Barley Stripe Mosaic Virus (BSMV), a disease of various cereals including barley and wheat, has been one of the successfully used viral RNA silencing tools in monocotyledonous plants for the last decade. In this study, we investigated several factors that play a significant role in VIGS. We tested the efficiency of silencing two genes simultaneously using the BSMV-induced gene silencing (IGS) system in detail. We found that two genes could be silenced simultaneously using BSMV-IGS. However, the silencing efficiency was found to be influenced by several factors including stability of the insert, temperature, and the accumulation of small viral RNAs from BSMV. The effect of these factors on VIGS system has been discussed

Transgene silencing and transgene-derived siRNA production in tobacco plants homozygous for an introduced AtMYB90 construct

Transgenic tobacco (Nicotiana tabacum) lines were engineered to ectopically over-express AtMYB90 (PAP2), an R2-R3 Myb gene associated with regulation of anthocyanin production in Arabidopsis thaliana. Independently transformed transgenic lines, Myb27 and Myb237, accumulated large quantities of anthocyanin, generating a dark purple phenotype in nearly all tissues. After self-fertilization, some progeny of the Myb27 line displayed an unexpected pigmentation pattern, with most leaves displaying large sectors of dramatically reduced anthocyanin production. The green-sectored 27Hmo plants were all found to be homozygous for the transgene and, despite a doubled transgene dosage, to have reduced levels of AtMYB90 mRNA. The observed reduction in anthocyanin pigmentation and AtMYB90 mRNA was phenotypically identical to the patterns seen in leaves systemically silenced for the AtMYB90 transgene, and was associated with the presence of AtMYB90-derived siRNA homologous to both strands of a portion of the AtMYB90 transcribed region. Activation of transgene silencing in the Myb27 line was triggered when the 35S::AtMYB90 transgene dosage was doubled, in both Myb27 homozygotes, and in plants containing one copy of each of the independently segregating Myb27 and Myb237 transgene loci. Mapping of sequenced siRNA molecules to the Myb27 TDNA (including flanking tobacco sequences) indicated that the 3' half of the AtMYB90 transcript is the primary target for siRNA associated silencing in both homozygous Myb27 plants and in systemically silenced tissues. The transgene within the Myb27 line was found to consist of a single, fully intact, copy of the AtMYB90 construct. Silencing appears to initiate in response to elevated levels of transgene mRNA (or an aberrant product thereof) present within a subset of leaf cells, followed by spread of the resulting small RNA to adjacent leaf tissues and subsequent amplification of siRNA production.Funding for this research is provided by the United States Department of Agriculture

A Spontaneous Dominant-Negative Mutation within a 35S::AtMYB90 Transgene Inhibits Flower Pigment Production in Tobacco

In part due to the ease of visual detection of phenotypic changes, anthocyanin pigment production has long been the target of genetic and molecular research in plants. Specific members of the large family of plant myb transcription factors have been found to play critical roles in regulating expression of anthocyanin biosynthetic genes and these genes continue to serve as important tools in dissecting the molecular mechanisms of plant gene regulation.A spontaneous mutation within the coding region of an Arabidopsis 35S::AtMYB90 transgene converted the activator of plant-wide anthocyanin production to a dominant-negative allele (PG-1) that inhibits normal pigment production within tobacco petals. Sequence analysis identified a single base change that created a premature nonsense codon, truncating the encoded myb protein. The resulting mutant protein lacks 78 amino acids from the wild type C-terminus and was confirmed as the source of the white-flower phenotype. A putative tobacco homolog of AtMYB90 (NtAN2) was isolated and found to be expressed in flower petals but not leaves of all tobacco plants tested. Using transgenic tobacco constitutively expressing the NtAN2 gene confirmed the NtAN2 protein as the likely target of PG-1-based inhibition of tobacco pigment production.Messenger RNA and anthocyanin analysis of PG-1Sh transgenic lines (and PG-1Sh x purple 35S::NtAN2 seedlings) support a model in which the mutant myb transgene product acts as a competitive inhibitor of the native tobacco NtAN2 protein. This finding is important to researchers in the field of plant transcription factor analysis, representing a potential outcome for experiments analyzing in vivo protein function in test transgenic systems that over-express or mutate plant transcription factors

Transgene Silencing and Transgene-Derived siRNA Production in Tobacco Plants Homozygous for an Introduced AtMYB90 Construct

Transgenic tobacco (Nicotiana tabacum) lines were engineered to ectopically over-express AtMYB90 (PAP2), an R2–R3 Myb gene associated with regulation of anthocyanin production in Arabidopsis thaliana. Independently transformed transgenic lines, Myb27 and Myb237, accumulated large quantities of anthocyanin, generating a dark purple phenotype in nearly all tissues. After self-fertilization, some progeny of the Myb27 line displayed an unexpected pigmentation pattern, with most leaves displaying large sectors of dramatically reduced anthocyanin production. The green-sectored 27Hmo plants were all found to be homozygous for the transgene and, despite a doubled transgene dosage, to have reduced levels of AtMYB90 mRNA. The observed reduction in anthocyanin pigmentation and AtMYB90 mRNA was phenotypically identical to the patterns seen in leaves systemically silenced for the AtMYB90 transgene, and was associated with the presence of AtMYB90-derived siRNA homologous to both strands of a portion of the AtMYB90 transcribed region. Activation of transgene silencing in the Myb27 line was triggered when the 35S::AtMYB90 transgene dosage was doubled, in both Myb27 homozygotes, and in plants containing one copy of each of the independently segregating Myb27 and Myb237 transgene loci. Mapping of sequenced siRNA molecules to the Myb27 TDNA (including flanking tobacco sequences) indicated that the 3′ half of the AtMYB90 transcript is the primary target for siRNA associated silencing in both homozygous Myb27 plants and in systemically silenced tissues. The transgene within the Myb27 line was found to consist of a single, fully intact, copy of the AtMYB90 construct. Silencing appears to initiate in response to elevated levels of transgene mRNA (or an aberrant product thereof) present within a subset of leaf cells, followed by spread of the resulting small RNA to adjacent leaf tissues and subsequent amplification of siRNA production

Evaluating the ability of the barley stripe mosaic virus-induced gene silencing system to simultaneously silence two wheat genes

Virus-induced gene silencing (VIGS) is an important tool for rapid assessment of gene function in plants. The ability of the Barley stripe mosaic virus (BSMV) VIGS system to simultaneously silence two genes was assessed by comparing the extent of down-regulation of the wheat PDS and SGT1 genes after wheat plants were infected with BSMV-VIGS constructs carrying 178bp fragments of the PDS and SGT1 genes linked in cis or after infection with single-gene silencing BSMV constructs carrying PDS or SGT1 alone or with a mixture of the single-gene constructs. Although significant variation occurred in these studies it was clearly demonstrated that two wheat genes can be silenced simultaneously by a single BSMV-VIGS construct

The Irish potato famine pathogen Phytophthora infestans translocates the CRN8 kinase into host plant cells

Phytopathogenic oomycetes, such as Phytophthora infestans, secrete an arsenal of effector proteins that modulate plant innate immunity to enable infection. We describe CRN8, a host-translocated effector of P. infestans that has kinase activity in planta. CRN8 is a modular protein of the CRN effector family. The C-terminus of CRN8 localizes to the host nucleus and triggers cell death when the protein is expressed in planta. Cell death induction by CRN8 is dependent on its localization to the plant nucleus, which requires a functional nuclear localization signal (NLS). The C-terminal sequence of CRN8 has similarity to a serine/threonine RD kinase domain. We demonstrated that CRN8 is a functional RD kinase and that its auto-phosphorylation is dependent on an intact catalytic site. Co-immunoprecipitation experiments revealed that CRN8 forms a dimer or multimer. Heterologous expression of CRN8 in planta resulted in enhanced virulence by P. infestans. In contrast, in planta expression of the dominant-negative CRN8R469A;D470A resulted in reduced P. infestans infection, further implicating CRN8 in virulence. Overall, our results indicate that similar to animal parasites, plant pathogens also translocate biochemically active kinase effectors inside host cells

Protein sequence allignment (ProbCon, [44]) of R2R3 Myb proteins demonstrated to produce a reduction in anthocyanin phenotypes when expressed in transgenic tobacco.

<p>Ectopic over-expression of the indicated Myb genes produces extensive purple pigmentation (P), white flower phenotype (W) or no phenotypic change (0). The R2 repeat is indicated as Red text and R3 repeat as Blue. Amino acid sequences that align in all proteins are boxed, differences between C1 andC1-I are shown as lower case. Sequences associated with Myb-bHLH interaction (L--R--RL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009917#pone.0009917-Grotewold2" target="_blank">[49]</a>, DL--R---L------L---R <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009917#pone.0009917-Zimmermann1" target="_blank">[50]</a>) are indicated above and below the aligned sequences. The amino acids encoded by the mRNA region of <i>AtMYB90</i> mRNA targeted by TAS4-siR81(-) are indicated by a grey box (<b>bold</b> indicates conservative amino acid differences between the <i>NtAN2</i> and <i>AtMYB90</i> sequences in that area). <b><i>Bold-italic</i></b> amino acids indicate the conserved C2 domain proposed to be important to <i>FaMYB1</i> repressor function <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009917#pone.0009917-Kranz1" target="_blank">[56]</a>. Due to a native nonsense mutation the protein sequence of the <i>AtMYB114</i> allele in the Columbia ecotype is predicted to end 32 amino acids upstream from the PG-1 nonsense mutation (at the F residue just prior to the TAS4-siR81 (-) grey boxed region).</p