Shyi-Dong Yeh

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.

Broad-Spectrum Resistance to Different Geographic Strains of Papaya ringspot virus in Coat Protein Gene Transgenic Papaya.

ABSTRACT Papaya ringspot virus (PRSV) is a major limiting factor for cultivation of papaya (Carica papaya) in tropical and subtropical areas throughout the world. Although the coat protein (CP) gene of PRSV has been transferred into papaya by particle bombardment and transgenic lines with high resistance to Hawaii strains have been obtained, they are susceptible to PRSV isolates outside of Hawaii. This strain-specific resistance limits the application of the transgenic lines in other areas of the world. In this investigation, the CP gene of a local strain isolated from Taiwan, designated PRSV YK, was transferred into papaya via Agrobacterium-mediated transformation. A total of 45 putative transgenic lines were obtained and the presence of the transgene in papaya was confirmed by polymerase chain reaction amplification. When the plants of transgenic lines were challenged with PRSV YK by mechanical inoculation, they showed different levels of resistance ranging from delay of symptom development to complete immunity. Molecular analysis of nine selected lines that exhibited different levels of resistance revealed that the expression level of the transgene is negatively correlated with the degree of resistance, suggesting that the resistance is manifested by a RNA-mediated mechanism. The segregation analysis showed that the transgene in the immune line 18-0-9 has an inheritance of two dominant loci and the other four highly resistant lines have a single dominant locus. Seven selected lines were tested further for resistance to three PRSV heterologous strains that originated in Hawaii, Thailand, and Mexico. Six of the seven lines showed varying degrees of resistance to the heterologous strains, and one line, 19-0-1, was immune not only to the homologous YK strain but also to the three heterologous strains. Thus, these CP-transgenic papaya lines with broad-spectrum resistance have great potential for use in Taiwan and other geographic areas to control PRSV.

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.

Translation of papaya ringspot virus RNA in vitro: Detection of a possible polyprotein that is processed for capsid protein, cylindrical-inclusion protein, and amorphous-inclusion protein

The genomic RNA of papaya ringspot virus (PRV), a member of the potyvirus group, was translated in a rabbit reticulocyte cell-free system as an approach to determining the translation strategy of the virus. The RNA directed synthesis of more than 20 distinct polypeptides ranging from apparent molecular weight of 26,000 (26K) to 220K. Antiserum to PRV capsid protein (CP) reacted with a subset of these polypeptides, including a 36K protein that comigrated with PRV CP during electrophoresis. Immunoprecipitation with antiserum to PRV cylindrical-inclusion protein (CIP) defined another set of polypeptides including 70K, 108K, 205K, and 220K proteins as major precipitates. The 70K protein comigrated with authentic CIP, and the 205K and 220K proteins were related to both CP and CIP. Immunoprecipitation with antiserum to PRV amorphous-inclusion protein (AIP) defined a unique set of polypeptides which contained a 112K protein as the major precipitate and 51K, 65K, and 86K proteins as minor precipitates. The 51K protein comigrated with authentic AIR A major product of 330K was observed when translation was done without the reducing agent, dithiothreitol. Immunological analyses and kinetic studies indicated that the 330K protein zone was related to the presumed CP, CIP, and AIP zones and 330K possibly is the common precursor for these viral proteins. The presence of a polyprotein of Mr corresponding to the entire coding capacity of the genomic RNA and its likely precursor relationship to the other polypeptides suggest that proteolytic processing is involved in the translation of PRV RNA.

Completion of the genome sequence of Watermelon silver mottle virus and utilization of degenerate primers for detecting tospoviruses in five serogroups.

ABSTRACT The nucleotide sequence of the L RNA of Watermelon silver mottle virus (WSMoV) was determined. Combined with the previous work on M and S RNAs, the whole genomic sequence of this member of the genus Tospovirus was completed. The L RNA is 8,917 nucleotides in length, with one large open reading frame encoding a translation product of 2,878 amino acids (331.8 kDa) on the viral complementary strand. The L protein shares amino acid identities of only 44.3 and 46.5% with Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus, respectively; but an amino acid identity of 91.3% with Peanut bud necrosis virus. Among the sequenced tospoviruses, L protein was the most conserved gene product, whereas the nonstructural S protein was generally the most variable. Comparison of the deduced L protein of WSMoV with those of other members of the family Bunyaviridae revealed that its amino acid sequence includes the reported conserved motifs of RNA-dependent RNA polymerases. To develop a method for detecting tospo-viruses by reverse transcription-polymerase chain reaction (RT-PCR), two pairs of degenerate primers were designed from conserved regions of the L genes and used to amplify the corresponding regions of the L genes from total RNAs extracted from plant tissues infected with five serologically distinct tospoviruses. The DNA fragments obtained were identified as those of tospoviruses by restriction enzyme digestion and DNA sequencing. For field samples, watermelon and wax gourd infected with WSMoV, and lisianthus infected with TSWV were also successfully detected by these two pairs of degenerate primers, with a sensitivity similar to N-gene-specific primers. The results indicated that the RT-PCR with the degenerate primers is a fast and reliable method for detecting tospoviruses in different serogroups.

Practices and perspective of control of papaya ringspot virus by cross protection

Papaya (Carica papaya L.) is widely grown in tropical and subtropical areas for its edible fruit and delicate taste. The plant grows fast and the fruit can be harvested 8–10 months after transplanting the tree in the field. It continues producing fruit for 2–3 years under normal conditions. The delicious and nutritious fruit contains a popular protease, papain, which helps to digest food for assimilation. The extensive adaption of this plant and wide acceptance of the fruit offer considerable promise for papaya as a commercial crop for local and export purpose. Like banana, pineapple, and mango, papaya is one of the important cash crops in the tropics and subtropics.

Divergence and conservation of the genomic RNAs of Taiwan and Hawaii strains of papaya ringspot potyvirus

SummaryThe complete nucleotide sequence of the genome of a Taiwan isolate of papaya ringspot potyvirus (PRSV YK) was determined from three overlapping cDNA clones and by direct RNA sequencing. Comparison was made with the reported Hawaii isolate of PRSV HA. Both genomes are 10 326 nucleotides long, excluding the poly(A)-tail. They encode a polyprotein of 3 344 amino acids with a 5′ leader of 85 nucleotides and a 3′ non-translated region of 209 nucleotides. The two genomes share an overall nucleotide identity of 83.4% and an amino acid identity of 90.6%. The 3′ non-translated regions show 92.3% identity. The first 23 nucleotides of the leaders are identical, while the remaining parts of the leaders only show 51.6% identity. The P1 protein genes of the two isolates are very different, with 70.9% nucleotide identity and 66.7% encoded amino acids identity. However, the other viral proteins of the two virus isolates are similar, with a 82.5–89.8% nucleotide identity of their genes and 91.2–97.6% amino acid identity, indicating that they are strains of the same potyvirus. Analysis of the ratios of nucleotide differences to the actual amino acid changes revealed that there are only 2.63 nucleotide changes for each amino acid change in the P1 protein, whereas for the other proteins 4.0–16.4 nucleotide changes are required for each amino acid replacement. The P1 protein has 58% of all the differences of polyprotein. The unusual variation in the leader sequences and the P1 proteins suggests that the two PRSV strains were derived from different evolutionary pathways in different geographic areas.

A Single Amino Acid of NIaPro of Papaya ringspot virus Determines Host Specificity for Infection of Papaya

Most strains of Papaya ringspot virus (PRSV) belong to type W, causing severe loss on cucurbits worldwide, or type P, devastating papaya in tropical areas. While the host range of PRSV W is limited to plants of the families Chenopodiaceae and Cucuribitaceae, PRSV P, in addition, infects plants of the family Caricaceae (papaya family). To investigate one or more viral genetic determinants for papaya infection, recombinant viruses were constructed between PRSV P-YK and PRSV W-CI. Host reactions to recombinant viruses indicated that the viral genomic region covering the C-terminal region (142 residues) of NIaVPg, full NIaPro, and N-terminal region (18 residues) of NIb, is critical for papaya infection. Sequence analysis of this region revealed residue variations at position 176 of NIaVPg and positions 27 and 205 of NIaPro between type P and W viruses. Host reactions to the constructed mutants indicated that the amino acid Lys27 of NIaPro determines the host-specificity of PRSV for papaya infection. Predicted three-dimensional structures of NIaPros of parental viruses suggested that Lys27 does not affect the protease activity of NIaPro. Recovery of the infected plants from certain papaya-infecting mutants implied involvement of other viral factors for enhancing virulence and adaptation of PRSV on papaya.

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.

Effects of carbenicillin and cefotaxime on callus growth and somatic embryogenesis from adventitious roots of papaya

Carbenicillin and cefotaxime, two antibiotics commonly used for excluding Agrobacterium tumefaciens during plant transformation, were tested for their bacteriostatic effects as well as for their effects on plant regeneration in adventitious root explants of papaya following co-culture with Agrobacterium. A washing step with sterilized distilled water two days after co-culture enhanced the bacteria-suppressing effects of antibiotics. Proliferation of Agrobacterium was completely suppressed in the medium containing 125mgl^(-1) carbenicillin or cefotaxime. Callus fresh weight increase was apparently enhanced in the media with higher concentrations of carbenicillin (250-500 mgl^(-1)), but was extremely inhibited in media with the same concentrations of cefotaxime. Higher percentages of somatic embryos were found in the medium with 125 mgl^(-1) carbenicillin or 250 mgl^(-1) cefotaxime; however larger numbers of somatic embryos from the individual callus were obtained in the medium with 125 mgl^(-1) carbenicillin than in the medium with 250 mgl^(-1) cefotaxime. Percentages of abnormal somatic embryos were lower in the medium with lower concentrations of carbenicillin (125-250 mgl^(-1)). Favorable conditions for use of the two antibiotics for suppressing bacteria growth and enhancing regeneration of papaya plantlets from adventitious roots are discussed.