A. Brunetti

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.

Molecular and physiological characterization of Italian isolates of Pyrenochaeta lycopersici

Corky root of tomato caused by Pyrenochaeta lycopersici is a disease of concern in Italy and for many tomato growing areas in the world. Isolates of the fungus were characterized at both the physiological and molecular level. The optimal in vitro growth temperature for all isolates was 23 degrees C. All Italian isolates of P. lycopersici showed similar RAPD and esterase banding patterns. No relevant polymorphisms were detected after enzymatic digestion of PCR-amplified ITS and IGS regions. The overall results indicate a low degree of genetic variability within a collection of 43 Italian isolates. These data are of interest in breeding programs for resistance against corky root of tomato and they provide useful information for the development of molecular diagnostic tools for the rapid identification and detection of P. lycopersici.

Kinase domain-targeted isolation of defense-related receptor-like kinases (RLK/Pelle) in Platanus × acerifolia: phylogenetic and structural analysis

BackgroundPlant receptor-like kinase (RLK/Pelle) family regulates growth and developmental processes and interaction with pathogens and symbionts.Platanaceae is one of the earliest branches of Eudicots temporally located before the split which gave rise to Rosids and Asterids. Thus investigations into the RLK family in Platanus can provide information on the evolution of this gene family in the land plants.Moreover RLKs are good candidates for finding genes that are able to confer resistance to Platanus pathogens.ResultsDegenerate oligonucleotide primers targeting the kinase domain of stress-related RLKs were used to isolate for the first time 111 RLK gene fragments in Platanus × acerifolia. Sequences were classified as candidates of the following subfamilies: CrRLK1L, LRR XII, WAK-like, and LRR X-BRI1 group. All the structural features typical of the RLK kinase domain were identified, including the non-RD motif which marks potential pathogen recognition receptors (PRRs). The LRR XII candidates, whose counterpart in Arabidopsis and rice comprises non-RD PRRs, were mostly non-RD kinases, suggesting a group of PRRs. Region-specific signatures of a relaxed purifying selection in the LRR XII candidates were also found, which is novel for plant RLK kinase domain and further supports the role of LRR XII candidates as PRRs. As we obtained CrRLK1L candidates using primers designed on Pto of tomato, we analysed the phylogenetic relationship between CrRLK1L and Pto-like of plant species. We thus classified all non-solanaceous Pto-like genes as CrRLK1L and highlighted for the first time the close phylogenetic vicinity between CrRLK1L and Pto group. The origins of Pto from CrRLK1L is proposed as an evolutionary mechanism.ConclusionsThe signatures of relaxed purifying selection highlight that a group of RLKs might have been involved in the expression of phenotypic plasticity and is thus a good candidate for investigations into pathogen resistance.Search of Pto-like genes in Platanus highlighted the close relationship between CrRLK1L and Pto group. It will be exciting to verify if sensu strictu Pto are present in taxonomic groups other than Solanaceae, in order to further clarify the evolutionary link with CrRLK1L.We obtained a first valuable resource useful for an in-depth study on stress perception systems.