Mario Tavazza

A viral protein suppresses RNA silencing and binds silencing-generated, 21- to 25-nucleotide double-stranded RNAs

Posttranscriptional gene silencing (PTGS) processes double‐stranded (ds) RNAs into 21–25 nucleotide (nt) RNA fragments that direct ribonucleases to target cognate mRNAs. In higher plants, PTGS also generates mobile signals conferring sequence‐specific silencing in distant organs. Since PTGS acts as an antiviral system in plants, successful virus infection requires evasion or suppression of gene silencing. Here we report that the 19 kDa protein (p19) of tombusviruses is a potent silencing suppressor that prevents the spread of mobile silencing signal. In vitro, p19 binds PTGS‐generated, 21–25 nt dsRNAs and 21‐nt synthetic dsRNAs with 2‐nt 3′ overhanging end(s), while it barely interacts with single‐stranded (ss) RNAs, long dsRNAs or blunt‐ended 21‐nt dsRNAs. We propose that p19 mediates silencing suppression by sequestering the PTGS‐generated 21–25 nt dsRNAs, thus depleting the specificity determinants of PTGS effector complexes. Moreover, the observation that p19‐expressing transgenic plants show altered leaf morphology might indicate that the p19‐targeted PTGS pathway is also important in the regulation of plant development.

Tomato Yellow Leaf Curl Sardinia Virus Rep-Derived Resistance to Homologous and Heterologous Geminiviruses Occurs by Different Mechanisms and Is Overcome if Virus-Mediated Transgene Silencing Is Activated

ABSTRACT The replication-associated protein (Rep) of geminiviruses is involved in several biological processes brought about by the presence of distinct functional domains. Recently, we have exploited the multifunctional character of the Tomato yellow leaf curl Sardinia virus (TYLCSV) Rep to develop a molecular interference strategy to impair TYLCSV infection. We showed that transgenic expression of its N-terminal 210 amino acids (Rep-210) confers resistance to the homologous virus by inhibiting viral transcription and replication. We have now used biochemical and transgenic approaches to carry out a fuller investigation of the molecular resistance mechanisms in transgenic plants expressing Rep-210. We show that Rep-210 confers resistance through two distinct molecular mechanisms, depending on the challenging virus. Resistance to the homologous virus is achieved by the ability of Rep-210 to tightly inhibit C1 gene transcription, while that to heterologous virus is due to the interacting property of the Rep-210 oligomerization domain. Furthermore, we present evidence that in Rep-210-expressing plants, the duration of resistance is related to the ability of the challenging virus to shut off transgene expression by a posttranscriptional homology-dependent gene silencing mechanism. A model of Rep-210-mediated geminivirus resistance that takes transgene- and virus-mediated mechanisms into account is proposed.

Hairpin RNA-mediated silencing of Plum pox virus P1 and HC-Pro genes for efficient and predictable resistance to the virus.

We report the application of the hairpin-mediated RNA silencing technology for obtaining resistance to Plum pox virus (PPV) infection in Nicotiana benthamiana plants. Four sequences, covering the P1 and silencing suppressor HC-Pro genes of an Italian PPV M isolate, were introduced into N. benthamiana plants as two inverted repeats separated by an intron sequence under the transcriptional control of the Cauliflower Mosaic Virus 35S promoter. In a leaf disk infection assay, 38 out of 40 T0 transgenic plants were resistant to PPV infection. Eight lines, 2 for each construct, randomly selected among the 38 resistant plants were further analysed. Two hundred forty eight out of 253 T1 transgenic plants were resistant to local and systemic PPV infection. All transgenic single locus lines were completely resistant. These data indicate that the RNA silencing of PPV P1/HCPro sequences results in an efficient and predictable PPV resistance, which may be utilized in obtaining stone fruit plants resistant to the devastating Sharka disease.

High Expression of Truncated Viral Rep Protein Confers Resistance to Tomato Yellow Leaf Curl Virus in Transgenic Tomato Plants

A truncated version of the C1 gene of tomato yellow leaf curl geminivirus (TYLCV), encoding the first 210 amino acids of the multifunctional Rep protein, was introduced by Agrobacterium transformation into Lycopersicon esculentum cv. Moneymaker plants under the transcriptional control of an enhanced cauliflower mosaic virus 35S promoter. One R0 plant (line 47) carrying the C1 gene in two loci (A and B) and accumulating the truncated Rep protein (T-Rep), was crossed with either a wild-type plant, or a C1 antisense plant (line 10). The wild type (wt) × 47 progeny were phenotypically homogeneous, contained either A or B locus, expressed high levels of T-Rep protein, had a “curled” phenotype, and were resistant to TYLCV when challenged either by agroinfection or by the vector Bemisia tabaci. In the 10 × 47 progeny, plants carrying only the sense gene behaved like the wt × 47 progeny, while those containing both sense and antisense transgenes did not accumulate the T-Rep protein, showed a normal phenotype, and...

Transgenically Expressed T-Rep of Tomato Yellow Leaf Curl Sardinia Virus Acts as a trans-Dominant-Negative Mutant, Inhibiting Viral Transcription and Replication

ABSTRACT We have previously shown that transgenic expression of a truncated C1 gene of Tomato yellow leaf curl Sardinia virus (TYLCSV), expressing the first 210 amino acids of the replication-associated protein (T-Rep) and potentially coexpressing the C4 protein, confers resistance to the homologous virus in Nicotiana benthamianaplants. In the present study we have investigated the role of T-Rep and C4 proteins in the resistance mechanism, analyzing changes in virus transcription and replication. Transgenic plants and protoplasts were challenged with TYLCSV and the related TYLCSV Murcia strain (TYLCSV-ES[1]). TYLCSV-resistant plants were susceptible to TYLCSV-ES[1]; moreover, TYLCSV but not TYLCSV-ES[1] replication was strongly inhibited in transgenic protoplasts as well as in wild-type (wt) protoplasts transiently expressing T-Rep but not the C4 protein. Viral circular single-stranded DNA (cssDNA) was usually undetectable in transgenically and transiently T-Rep-expressing protoplasts, while viral DNAs migrating more slowly than the cssDNA were observed. Biochemical studies showed that these DNAs were partial duplexes with the minus strand incomplete. Interestingly, similar viral DNA forms were also found at early stages of TYLCSV replication in wt N. benthamiana protoplasts. Transgenically expressed T-Rep repressed the transcription of the GUS reporter gene up to 300-fold when fused to the homologous (TYLCSV) but not to the heterologous (TYLCSV-ES[1]) C1 promoter. Similarly, transiently expressed T-Rep but not C4 protein strongly repressed GUS transcription when fused to the C1 promoter of TYLCSV. A model of T-Rep interference with TYLCSV transcription-replication is proposed.

Genetic transformation of potato (Solanum tuberosum): An efficient method to obtain transgenic plants

A quick procedure for efficient transformation of potato (cv. Desiree) has been devised. Leaf disc have been inoculated with an Agrobacterium tumefaciens harbouring a Ti plasmid derived binary vector. Transformed shoots carrying the neomycin phosphotransferase gene were regenerated using a feeder layer technique after 4 weeks on selective medium containing kanamycin. Transgenic plants obtained in large numbers appeared phenotypically normal and expressed the NPT II gene. The present work points out that culture conditions are fundamental to obtain the highest transformation efficiency.

Nucleotide sequence, genomic organization and synthesis of infectious transcripts from a full-length clone of artichoke mottle crinkle virus.

The complete nucleotide sequence of the genome of artichoke mottle crinkle virus (AMCV), a member of the tombusvirus group, has been determined. The genome is 4790 nucleotides (nt) in length. A full-length cDNA of the AMCV genome has been cloned in pUC9 downstream of the T7 RNA polymerase promoter. Transcripts were infective when inoculated onto Nicotiana clevelandii and N. benthamiana plants. The AMCV genome contains five open reading frames (ORFs). The first ORF from the 5 terminus (ORF1) encodes a protein with a predicted M(r) of 33K. ORF2 extends through the amber termination codon of ORF1 to yield a polypeptide of predicted M(r) 92K and which is the putative RNA-dependent RNA polymerase. ORF3 codes for the coat protein (41K). Two nested ORFs in different reading frames (ORFs 4 and 5) code for a 22K and a 19K polypeptide respectively. Sequence homologies suggest that the 22K protein could be involved in cell-to-cell movement of virus. ORFs 3, 4 and 5 are translated from two 3 coterminal subgenomic (sg) RNAs, the 5 termini of which have been mapped. The two sg RNAs are 2155 (sg1) and 934 (sg2) nt in length. ORF3 is expressed from sg1 RNA whereas ORFs 4 and 5 are potentially expressed from sg2 RNA. Time course experiments with Cynara scolymus protoplasts indicate that during AMCV infection both positive and negative strands of genomic and sg RNAs are produced and that sg2 RNA is produced before and at a higher level than sg1 RNA.

A cautionary note on pathogen-derived sequences

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Silencing of Plum pox virus 5′UTR/P1 sequence confers resistance to a wide range of PPV strains

An effective disease-control strategy should protect the host from the major economically important and geographically widespread variants of a pathogen. Plum pox virus (PPV) is the causal agent of sharka, the most devastating viral disease of Prunus species. We have shown previously that the hairpin RNA expression driven by h-UTR/P1, h-P1/HCPro, h-HCPro and h-HCPro/P3 constructs, derived from the PPV-M ISPaVe44 isolate, confers resistance to the homologous virus in Nicotiana benthamiana plants. Since the production of transgenic stone fruits and their evaluation for PPV resistance would take several years, the ISPaVe44-resistant plant lines were used to evaluate which construct would be the best candidate to be transferred to Prunus elite cultivars. To do that, nine PPV isolates of the D, M, Rec, EA and C strains originally collected from five Prunus species in different geographical areas, were typed by sequencing and used to challenge the transgenic N. benthamiana lines; 464 out of 464 virus-inoculated plants of lines h-UTR/P1, h-HCPro and h-HCPro/P3 showed complete and long-lasting resistance to the seven PPV isolates of D, M and Rec strains. Moreover, the h-UTR/P1 plants were also fully resistant to PPV-C and -EA isolates. Our data suggest that the h-UTR/P1 construct is of particular practical interest to obtain stone fruit plants resistant to the sharka disease.

Genetic Transformation of Nicotiana clevelandii Using a Ti Plasmid Derived Vector

Summary A procedure for the rapid production of transgenic shoots and F 1 progeny from Nicotiana clevelandii is described. Leaf discs were co-cultivated with a disarmed strain of Agrobacterium tumefaciens harbouring a binary vector system utilizing the neomycin phosphotransferase II as reporter gene. Inoculated leaf discs produced transgenic shoots at high frequency (50 %) after culturing for about three weeks on a regeneration medium which also contained 150 mg/l Kanamycin. The transgenic plants directly initiated flowering in vitro , producing a F 1 transgenic progeny. The life cycle was completed in approx. 4 months (from inoculation to F 1 progeny). Molecular analysis of the neomycin phosphotransferase marker confirmed the insertion, the expression and the transmission of the transgene to the F 1 progeny. Due to the wide use of N. clevelandii as a propagation host for most viruses the technique is of potential use in studies aimed at conferring engineered protection against viruses.