Shih-Shun Lin

Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method

Collective efforts of several laboratories in the past two decades have resulted in the development of various methods for Agrobacterium tumefaciens–mediated transformation of Arabidopsis thaliana. Among these, the floral dip method is the most facile protocol and widely used for producing transgenic Arabidopsis plants. In this method, transformation of female gametes is accomplished by simply dipping developing Arabidopsis inflorescences for a few seconds into a 5% sucrose solution containing 0.01–0.05% (vol/vol) Silwet L-77 and resuspended Agrobacterium cells carrying the genes to be transferred. Treated plants are allowed to set seed which are then plated on a selective medium to screen for transformants. A transformation frequency of at least 1% can be routinely obtained and a minimum of several hundred independent transgenic lines generated from just two pots of infiltrated plants (20–30 plants per pot) within 2–3 months. Here, we describe the protocol routinely used in our laboratory for the floral dip method for Arabidopsis transformation. Transgenic Arabidopsis plants can be obtained in approximately 3 months.

Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance

Plant microRNAs (miRNAs) regulate the abundance of target mRNAs by guiding their cleavage at the sequence complementary region. We have modified an Arabidopsis thaliana miR159 precursor to express artificial miRNAs (amiRNAs) targeting viral mRNA sequences encoding two gene silencing suppressors, P69 of turnip yellow mosaic virus (TYMV) and HC-Pro of turnip mosaic virus (TuMV). Production of these amiRNAs requires A. thaliana DICER-like protein 1. Transgenic A. thaliana plants expressing amiR-P69159 and amiR-HC-Pro159 are specifically resistant to TYMV and TuMV, respectively. Expression of amiR-TuCP159 targeting TuMV coat protein sequences also confers specific TuMV resistance. However, transgenic plants that express both amiR-P69159 and amiR-HC-Pro159 from a dimeric pre-amiR-P69159/amiR-HC-Pro159 transgene are resistant to both viruses. The virus resistance trait is displayed at the cell level and is hereditable. More important, the resistance trait is maintained at 15 °C, a temperature that compromises small interfering RNA–mediated gene silencing. The amiRNA-mediated approach should have broad applicability for engineering multiple virus resistance in crop plants.

Molecular Evolution of a Viral Non-Coding Sequence under the Selective Pressure of amiRNA-Mediated Silencing

Plant microRNAs (miRNA) guide cleavage of target mRNAs by DICER-like proteins, thereby reducing mRNA abundance. Native precursor miRNAs can be redesigned to target RNAs of interest, and one application of such artificial microRNA (amiRNA) technology is to generate plants resistant to pathogenic viruses. Transgenic Arabidopsis plants expressing amiRNAs designed to target the genome of two unrelated viruses were resistant, in a highly specific manner, to the appropriate virus. Here, we pursued two different goals. First, we confirmed that the 21-nt target site of viral RNAs is both necessary and sufficient for resistance. Second, we studied the evolutionary stability of amiRNA-mediated resistance against a genetically plastic RNA virus, TuMV. To dissociate selective pressures acting upon protein function from those acting at the RNA level, we constructed a chimeric TuMV harboring a 21-nt, amiRNA target site in a non-essential region. In the first set of experiments designed to assess the likelihood of resistance breakdown, we explored the effect of single nucleotide mutation within the target 21-nt on the ability of mutant viruses to successfully infect amiRNA-expressing plants. We found non-equivalency of the target nucleotides, which can be divided into three categories depending on their impact in virus pathogenicity. In the second set of experiments, we investigated the evolution of the virus mutants in amiRNA-expressing plants. The most common outcome was the deletion of the target. However, when the 21-nt target was retained, viruses accumulated additional substitutions on it, further reducing the binding/cleavage ability of the amiRNA. The pattern of substitutions within the viral target was largely dominated by G to A and C to U transitions.

RING1 E3 ligase localizes to plasma membrane lipid rafts to trigger FB1‐induced programmed cell death in Arabidopsis

Ubiquitination plays important roles in plant development, including programmed cell death. Here, we characterize a novel membrane-bound RING motif protein, encoded by RING1, that is expressed at a low level in all Arabidopsis tissues but can be upregulated by fumonisin B1 (FB1) treatment and pathogen infection. RING1 displays E3 ubiquitin ligase activity in vitro, which is dependent on the integrity of the RING motif. GFP fusion protein localization and cell fractionation experiments show that this E3 ligase is associated with the lipid rafts of plasma membranes. Knock-down of RING1 transcripts using artificial microRNA (amiR-R1(159)) leads to FB1 hyposensitivity, but overexpression of RING1 confers hypersensitivity. Additionally, expression of the pathogenesis-related 1 (PR-1) gene is lower and delayed in amiR-R1(159) plants compared with wild-type and RING1-overexpressing plants. The FB1 hyposensitivity of amiR-R1(159) plants can be rescued by expression of cleavage-resistant RING1mut transcripts. Our results suggest that RING1 acts as a signal from the plasma membrane lipid rafts to trigger the FB1-induced plant programmed cell death pathway.

Draft genome sequences of four strains of Vibrio parahaemolyticus, three of which cause early mortality syndrome/acute hepatopancreatic necrosis disease in shrimp in China and Thailand

ABSTRACT We sequenced four Vibrio parahaemolyticus strains, three of which caused serious acute hepatopancreatic necrosis disease. Sequence analysis of the virulent strains revealed not only genes related to cholera toxin and the type IV pilus/type IV secretion system but also a unique, previously unreported, large extrachromosomal plasmid that encodes a homolog to the insecticidal Photorhabdus insect-related binary toxin PirAB.

Multiple artificial microRNAs targeting conserved motifs of the replicase gene confer robust transgenic resistance to negative-sense single-stranded RNA plant virus

MicroRNAs (miRNAs) regulate the abundance of target mRNAs by guiding cleavage at sequence complementary regions. In this study, artificial miRNAs (amiRNAs) targeting conserved motifs of the L (replicase) gene of Watermelon silver mottle virus (WSMoV) were constructed using Arabidopsis pre-miRNA159a as the backbone. The constructs included six single amiRNAs targeting motifs A, B1, B2, C, D of E, and two triple amiRNAs targeting motifs AB1E or B2DC. Processing of pre-amiRNAs was confirmed by agro-infiltration, and transgenic Nicotiana benthamiana plants expressing each amiRNA were generated. Single amiRNA transgenic lines expressing amiR-LB2 or amiR-LD showed resistance to WSMoV by delaying symptom development. Triple amiRNA lines expressing amiR-LB2, amiR-LD and amiR-LC provided complete resistance against WSMoV, with no indication of infection 28 days after inoculation. Resistance levels were positively correlated with amiRNA expression levels in these single and triple amiRNA lines. The triple amiR-LAB1E line did not provide resistance to WSMoV. Similarly, the poorly expressed amiR-LC and amiR-LE lines did not provide resistance to WSMoV. The amiR-LA- and amiR-LB1-expressing lines were susceptible to WSMoV, and their additional susceptibility to the heterologous Turnip mosaic virus harbouring individual target sequences indicated that these two amiRNAs have no effect in vivo. Transgenic lines expressing amiR-LB2 exhibited delayed symptoms after challenge with Peanut bud necrosis virus having a single mismatch in the target site. Overall, our results indicate that two amiRNAs, amiR-LB2 and amiR-LD, of the six designed amiRNAs confer moderate resistance against WSMoV, and the triple construct including the two amiRNAs provides complete resistance.

Tempo and Mode of Plant RNA Virus Escape from RNA Interference-Mediated Resistance

ABSTRACT A biotechnological application of artificial microRNAs (amiRs) is the generation of plants that are resistant to virus infection. This resistance has proven to be highly effective and sequence specific. However, before these transgenic plants can be deployed in the field, it is important to evaluate the likelihood of the emergence of resistance-breaking mutants. Two issues are of particular interest: (i) whether such mutants can arise in nontransgenic plants that may act as reservoirs and (ii) whether a suboptimal expression level of the transgene, resulting in subinhibitory concentrations of the amiR, would favor the emergence of escape mutants. To address the first issue, we experimentally evolved independent lineages of Turnip mosaic virus (TuMV) (family Potyviridae) in fully susceptible wild-type Arabidopsis thaliana plants and then simulated the spillover of the evolving virus to fully resistant A. thaliana transgenic plants. To address the second issue, the evolution phase took place with transgenic plants that expressed the amiR at subinhibitory concentrations. Our results show that TuMV populations replicating in susceptible hosts accumulated resistance-breaking alleles that resulted in the overcoming of the resistance of fully resistant plants. The rate at which resistance was broken was 7 times higher for TuMV populations that experienced subinhibitory concentrations of the antiviral amiR. A molecular characterization of escape alleles showed that they all contained at least one nucleotide substitution in the target sequence, generally a transition of the G-to-A and C-to-U types, with many instances of convergent molecular evolution. To better understand the viral population dynamics taking place within each host, as well as to evaluate relevant population genetic parameters, we performed in silico simulations of the experiments. Together, our results contribute to the rational management of amiR-based antiviral resistance in plants.

Modifications of the Helper Component-Protease of Zucchini yellow mosaic virus for Generation of Attenuated Mutants for Cross Protection Against Severe Infection

ABSTRACT A nonpathogenic mild strain is essential for control of plant viruses by cross protection. Three amino acid changes, Arg(180)-->Ile(180) (GA mutation), Phe(205)-->Leu(205) (GB mutation), and Glu(396)-->Asn(396) (GC mutation), of the conserved motifs of the helper component-protease (HC-Pro) of a severe strain TW-TN3 of Zucchini yellow mosaic virus (ZYMV), a member of the genus Potyvirus, were generated from an infectious cDNA clone that carried a green fluorescent protein reporter. The infectivity of individual mutants containing single, double, or triple mutations was assayed on local and systemic hosts. On Chenopodium quinoa plants, the GB mutant induced necrotic lesions; the GA, GC, and GBC mutants induced chlorotic spots; and the GAB and GAC mutants induced local infection only visualized by fluorescence microscopy. On squash plants, the GA, GB, GC, and GBC mutants caused milder mosaic; the GAC mutant induced slight leaf mottling followed by recovering; and the GAB mutant did not induce conspicuous symptoms. Also, the GAC mutant, but not the GAB mutant, conferred complete cross protection against the parental virus carrying a mite allergen as a reporter. When tested on transgene-silenced transgenic squash, the ability of posttranscriptional gene silencing suppression of the mutated HC-Pro of GAC was not significantly affected. We concluded that the mutations of the HC-Pro of ZYMV reduce the degrees of pathogenicity on squash and also abolish the ability for eliciting the hypersensitive reaction on C. quinoa, and that the mutant GAC is a useful mild strain for cross protection.

Discriminating mutations of HC-Pro of zucchini yellow mosaic virus with differential effects on small RNA pathways involved in viral pathogenicity and symptom development.

Helper component-proteinase (HC-Pro), the gene-silencing suppressor of Potyvirus spp., interferes with microRNA (miRNA) and short-interfering RNA (siRNA) pathways. Our previous studies showed that three mutations of highly conserved amino acids of HC-Pro, R(180)I (mutation A), F(205)L (B), and E(396)N (C), of Zucchini yellow mosaic virus (ZYMV) affect symptom severity and viral pathogenicity. The mutant ZYMV GAC (ZGAC) with double mutations, R(180)I/E(396)N, induces transient leaf mottling in host plants followed by recovery. This mutant confers complete cross protection against subsequent infection by the parental ZYMV (ZG) strain. Here, we sought to obtain molecular evidence on the roles of the three highly conserved amino acids of HC-Pro in miRNA and siRNA pathways using transgenic Arabidopsis plants expressing comparable levels of wild-type and mutant HC-Pro proteins. We demonstrated that amino acid residues 180, 205, and 396 of HC-Pro are critical for suppression of miRNA, trans-acting siRNA (ta-siRNA), and virus-induced gene silencing (VIGS) pathways but not for sense-post transcriptional gene silencing (s-PTGS). Because the HC-Pro double mutant (R(180)I/E(396)N) does not interfere with miRNA and ta-siRNA pathways, the ZGAC mutant virus elicits only attenuated symptoms. Furthermore, the recovery seen on ZGAC-infected plants likely results from the weak VIGS suppression by the HC-Pro double AC mutant. Thus, through manipulating these three conserved amino acids on HC-Pro, symptom severity of diseases caused by Potyvirus spp. can be modulated to generate useful cross protectants for field application. Although some of our mutated HC-Pro proteins do not interfere with miRNA and ta-siRNA pathways, they still retain the ability to suppress s-PTGS.

Strategies and mechanisms of plant virus resistance

Virus-induced diseases are responsible for major crop losses worldwide. A better understanding of plant defense mechanisms would lead to the development of novel strategies for effective plant protection. Early protein-based approaches relied mostly on the expression of transgenic coat protein (CP) to block the progression of the virus infectious process. Other strategies exploit the plant’s innate defense mechanisms to combat invading viral pathogens. For example, the RNA-based resistance makes use of the plant post-transcriptional gene silencing (PTGS) mechanism to degrade viral RNAs. In cross-protection the prior inoculation with a mild viral strain confers resistance against a severe strain. Although the molecular detail of cross-protection is not fully understood, it is likely to be comprised of both protein- and RNA-based mechanisms, as well as some other unknown processes. In this review article we compare the benefits and challenges of these different viral-resistance approaches. Furthermore, we discuss the development of a new approach based on the plant’s miRNA pathway. Artificial miRNAs with sequences complementary to viral sequences have been successfully used to generate virus resistance. This novel anti-viral strategy, which has the advantage of reducing possible bio-safety risks associated with protein- and RNA-based strategies, is a first step toward designing environmentally friendly virus resistance in transgenic crops.