Marina Barba

Historical Perspective, Development and Applications of Next-Generation Sequencing in Plant Virology

Next-generation high throughput sequencing technologies became available at the onset of the 21st century. They provide a highly efficient, rapid, and low cost DNA sequencing platform beyond the reach of the standard and traditional DNA sequencing technologies developed in the late 1970s. They are continually improved to become faster, more efficient and cheaper. They have been used in many fields of biology since 2004. In 2009, next-generation sequencing (NGS) technologies began to be applied to several areas of plant virology including virus/viroid genome sequencing, discovery and detection, ecology and epidemiology, replication and transcription. Identification and characterization of known and unknown viruses and/or viroids in infected plants are currently among the most successful applications of these technologies. It is expected that NGS will play very significant roles in many research and non-research areas of plant virology.

Next-Generation Sequencing and Genome Editing in Plant Virology

Next-generation sequencing (NGS) has been applied to plant virology since 2009. NGS provides highly efficient, rapid, low cost DNA, or RNA high-throughput sequencing of the genomes of plant viruses and viroids and of the specific small RNAs generated during the infection process. These small RNAs, which cover frequently the whole genome of the infectious agent, are 21–24 nt long and are known as vsRNAs for viruses and vd-sRNAs for viroids. NGS has been used in a number of studies in plant virology including, but not limited to, discovery of novel viruses and viroids as well as detection and identification of those pathogens already known, analysis of genome diversity and evolution, and study of pathogen epidemiology. The genome engineering editing method, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been successfully used recently to engineer resistance to DNA geminiviruses (family, Geminiviridae) by targeting different viral genome sequences in infected Nicotiana benthamiana or Arabidopsis plants. The DNA viruses targeted include tomato yellow leaf curl virus and merremia mosaic virus (begomovirus); beet curly top virus and beet severe curly top virus (curtovirus); and bean yellow dwarf virus (mastrevirus). The technique has also been used against the RNA viruses zucchini yellow mosaic virus, papaya ringspot virus and turnip mosaic virus (potyvirus) and cucumber vein yellowing virus (ipomovirus, family, Potyviridae) by targeting the translation initiation genes eIF4E in cucumber or Arabidopsis plants. From these recent advances of major importance, it is expected that NGS and CRISPR-Cas technologies will play a significant role in the very near future in advancing the field of plant virology and connecting it with other related fields of biology.

Extreme resistance to cucumber mosaic virus (CMV) in transgenic tomato expressing one or two viral coat proteins

For the production of broad commercial resistance to cucumber mosaic virus (CMV) infection, tomato plants were transformed with a combination of two coat protein (CP) genes, representing both subgroups of CMV. The CP genes were cloned from the CMV-D strain and Italian CMV isolates (CMV-22 of subgroup I and CMV-PG of subgroup II) which have been shown to produce severe disease symptoms. Four plant transformation vectors were constructed: pMON18774 and pMON18775 (CMV-D CP), pMON18831 (CMV-PG CP) and pMON18833 (CMV-22 CP and CMV-PG CP). Transformed R0 plants were produced and lines were selected based on the combination of three traits: CMV CP expression at the R0 stage, resistance to CMV (subgroup I and/or II) infection in growth chamber tests in R1 expressing plants, and single transgene copy, based on R1 segregation. The results indicate that all four vector constructs generated plants with extremely high resistant to CMV infection. The single and double gene vector construct produced plants with broad resistance against strains of CMV from both subgroups I and II at high frequency. The engineered resistance is of practical value and will be applied for major Italian tomato varieties.

Viroids and RNA silencing: Mechanism, role in viroid pathogenicity and development of viroid-resistant plants

Viroids are autonomously replicating, small single-stranded circular RNA pathogens that do not code for proteins and may cause diseases in infected, susceptible plants. They have the ability to induce both RNA-mediated transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS), or RNA silencing, in infected plants. Their induced RNA silencing has also been demonstrated in a wheat germ extract system. A possible role of RNA silencing in viroid pathogenicity and evolution has been discussed. It is suggested that RNA silencing can be employed to engineer plants for viroid resistance and attempts to produce these plants have been also discussed.

Resistance of transgenic tomato to cucumber mosaic cucumovirus under field conditions

Since the summer of 1993, transgenic tomato plants expressing the coat protein (CP) genes of cucumber mosaic cucumovirus have been tested under field conditions to assess the level of resistance and agronomic performance. Trials were performed in different areas in Italy and the target virus in the majority of tests was spread naturally by the indigenous aphid populations. Twenty-three homozygous lines of variety UC82B, transformed to contain four different CP genes of CMV, were evaluated. The lines were preselected for CP expression, single gene copy, and virus resistance in growth chamber experiments. In general, CMV resistance was confirmed under field conditions though resistance in the field was less effective than what was observed in growth chamber experiments. The resistance observed in multi-year and multi-location experiments is of commercial value for several of the most resistant lines. Engineered resistance upon transfer to Italian varieties by breeding or direct transformation will be used in tomato production in Italy or elsewhere.

Molecular Diagnosis of "Chrysanthemum stunt viroid" for Routine Indexing

In 2002, a two-year study was started to check for Chrysanthemum stunt viroid (CSVd) during the production and selective breeding of new chrysanthemum varieties in a central-Italy flower-growing farm. Two molecular techniques, one-tube RT-PCR and tissue printing for hybridization assays, were improved for their effectiveness in viroid detection at different stages of plant selection. Both molecular techniques proved sensitive, reliable and easy to apply in a programme of routine indexing for the production of new and healthy chrysanthemum varieties.

Optimization and improvement of oligonucleotide microarray-based detection of tomato viruses and pospiviroids

Tomato (Solanum lycopersicum L.) is a vegetable crop which is affected by many viruses and several viroids, causing significant economic loss. Their detection and identification is of critical importance for plant protection and quarantine and certification programs. The potential was examined of an array based on the Combimatrix platform for the detection of 37 viruses belonging to 13 families, one of which is unassigned, together with six pospiviroid species, genus Pospiviroid, family Pospiviroidae. More than 470 oligonucleotide probes (40-mer) were selected for the microarray diagnostic technique developed in this investigation. Most of the virus probes were highly specific and were able to identify tomato viruses. Most pospiviroid probes, however, were non-specific in terms of species, but were specific at the genus level as they hybridized to members of the genus Pospiviroid. Only one probe of the Tomato apical stem viroid was species specific. The repeatability and specificity of the Combimatrix method showed that it can be considered for routine diagnostic use in suspected tomato germplasm since it detected 37 viruses and one pospiviroid at the species level and 5-6 pospiviroids at the genus level. The estimated cost for testing of a single tomato virus is similar to or less than the cost of using ELISA.

Occurrence of dapple fruit of plum in Italy

In the course of a field survey of viroids of stone fruit trees in southern Italy, we encountered some particular fruit symptoms in plum cv. Florentia and Sorriso di Primavera, which resembled the symptoms of a disease described in Japan in 1989 and was named dapple fruit disease. Symptoms consisted mainly of discolored spots on the fruit skins, and led to loss of market value. Hop stunt viroid (HSVd) was found in all symptomatic trees, but not in any symptomless samples. No other viroid was found in these trees. All samples were also checked for the more common viruses affecting plum. Apple chlorotic leaf spot trichovirus (ACLSV) was detected in both symptomatic and symptomless trees in similar percentages but no other viruses were found in any of the samples. These results provide evidence for the belief that HSVd is the cause of dapple fruit. This is the first report of dapple fruit in Italy.