Immunity to tomato yellow leaf curl virus in transgenic tomato is associated with accumulation of transgene small RNA.

Gene silencing is a natural defense response of plants against invading RNA and DNA viruses. The RNA post-transcriptional silencing system has been commonly utilized to generate transgenic crop plants that are "immune" to plant virus infection. Here, we applied this approach against the devastating DNA virus tomato yellow leaf curl virus (TYLCV) in its host tomato (Solanum lycopersicum L.). To generate broad resistance to a number of different TYLCV viruses, three conserved sequences (the intergenic region [NCR], V1-V2 and C1-C2 genes) from the genome of the severe virus (TYLCV) were synthesized as a single insert and cloned into a hairpin configuration in a binary vector, which was used to transform TYLCV-susceptible tomato plants. Eight of 28 independent transgenic tomato lines exhibited immunity to TYLCV-Is and to TYLCV-Mld, but not to tomato yellow leaf curl Sardinia virus, which shares relatively low sequence homology with the transgene. In addition, a marker-free (nptII-deleted) transgenic tomato line was generated for the first time by Agrobacterium-mediated transformation without antibiotic selection, followed by screening of 1180 regenerated shoots by whitefly-mediated TYLCV inoculation. Resistant lines showed a high level of transgene-siRNA (t-siRNA) accumulation (22% of total small RNA) with dominant sizes of 21 nt (73%) and 22 nt (22%). The t-siRNA displayed hot-spot distribution ("peaks") along the transgene, with different distribution patterns than the viral-siRNA peaks observed in TYLCV-infected tomato. A grafting experiment demonstrated the mobility of 0.04% of the t-siRNA from transgenic rootstock to non-transformed scion, even though scion resistance against TYLCV was not achieved.

Frequent migration of introduced cucurbit-infecting begomoviruses among Middle Eastern countries.

BACKGROUND: In the early 2000s, two cucurbit-infecting begomoviruses were introduced into the eastern Mediterranean basin: the Old World Squash leaf curl virus (SLCV) and the New World Watermelon chlorotic stunt virus (WmCSV). These viruses have been emerging in parallel over the last decade in Egypt, Israel, Jordan, Lebanon and Palestine. METHODS: We explored this unique situation by assessing the diversity and biogeography of the DNA-A component of SLCV and WmCSV in these five countries. RESULTS: There was fairly low sequence variation in both begomovirus species (SLCV π = 0.0077; WmCSV π = 0.0066). Both viruses may have been introduced only once into the eastern Mediterranean basin, but once established, these viruses readily moved across country boundaries. SLCV has been introduced at least twice into each of all five countries based on the absence of monophyletic clades. Similarly, WmCSV has been introduced multiple times into Jordan, Israel and Palestine. CONCLUSIONS: We predict that uncontrolled movement of whiteflies among countries in this region will continue to cause SLCV and WmCSV migration, preventing strong genetic differentiation of these viruses among these countries.

Exploiting Virus Infection to Protect Plants from Abiotic Stresses: Tomato Protection by a Begomovirus.

Tomato cultivation is threatened by environmental stresses (e.g., heat, drought) and by viral infection (mainly viruses belonging to the tomato yellow leaf curl virus family-TYLCVs). Unlike many RNA viruses, TYLCV infection does not induce a hypersensitive response and cell death in tomato plants. To ensure a successful infection, TYLCV preserves a suitable cellular environment where it can reproduce. Infected plants experience a mild stress, undergo adaptation and become partially "ready" to exposure to other environmental stresses. Plant wilting and cessation of growth caused by heat and drought is suppressed by TYLCV infection, mainly by down-regulating the heat shock transcription factors, HSFA1, HSFA2, HSFB1 and consequently, the expression of HSF-regulated stress genes. In particular, TYLCV captures HSFA2 by inducing protein complexes and aggregates, thus attenuating an acute stress response, which otherwise causes plant death. Viral infection mitigates the increase in stress-induced metabolites, such as carbohydrates and amino acids, and leads to their reallocation from shoots to roots. Under high temperatures and water deficit, TYLCV induces plant cellular homeostasis, promoting host survival. Thus, this virus-plant interaction is beneficial for both partners.

Interplay between abiotic (drought) and biotic (virus) stresses in tomato plants.

With climate warming, drought becomes a vital challenge for agriculture. Extended drought periods affect plant-pathogen interactions. We demonstrate an interplay in tomato between drought and infection with tomato yellow leaf curl virus (TYLCV). Infected plants became more tolerant to drought, showing plant readiness to water scarcity by reducing metabolic activity in leaves and increasing it in roots. Reallocation of osmolytes, such as carbohydrates and amino acids, from shoots to roots suggested a role of roots in protecting infected tomatoes against drought. To avoid an acute response possibly lethal for the host organism, TYLCV down-regulated the drought-induced activation of stress response proteins and metabolites. Simultaneously, TYLCV promoted the stabilization of osmoprotectants' patterns and water balance parameters, resulting in the development of buffering conditions in infected plants subjected to prolonged stress. Drought-dependent decline of TYLCV amounts was correlated with HSFA1-controlled activation of autophagy, mostly in the roots. The tomato response to combined drought and TYLCV infection points to a mutual interaction between the plant host and its viral pathogen.

Tomato Yellow Leaf Curl Virus (TYLCV) Promotes Plant Tolerance to Drought.

A growing body of research points to a positive interplay between viruses and plants. Tomato yellow curl virus (TYLCV) is able to protect tomato host plants against extreme drought. To envisage the use of virus protective capacity in agriculture, TYLCV-resistant tomato lines have to be infected first with the virus before planting. Such virus-resistant tomato plants contain virus amounts that do not cause disease symptoms, growth inhibition, or yield loss, but are sufficient to modify the metabolism of the plant, resulting in improved tolerance to drought. This phenomenon is based on the TYLCV-dependent stabilization of amounts of key osmoprotectants induced by drought (soluble sugars, amino acids, and proteins). Although in infected TYLCV-susceptible tomatoes, stress markers also show an enhanced stability, in infected TYLCV-resistant plants, water balance and osmolyte homeostasis reach particularly high levels. These tomato plants survive long periods of time during water withholding. However, after recovery to normal irrigation, they produce fruits which are not exposed to drought, similarly to the control plants. Using these features, it might be possible to cultivate TYLCV-resistant plants during seasons characterized by water scarcity.

Oligonucleotide microarray-based detection and genotyping of Plum pox virus.

Plum pox virus (PPV) is the most damaging viral pathogen of stone fruits. The detection and identification of its strains are therefore of critical importance to plant quarantine and certification programs. Existing methods to screen strains of PPV suffer from significant limitations such as the simultaneous detection and genotyping of several strains of PPV in samples infected with different isolates of the virus. A genomic strategy for PPV screening based on the viral nucleotide sequence was developed to enable the detection and genotyping of the virus from infected plant tissue or biological samples. The basis of this approach is a long 70-mer oligonucleotide DNA microarray capable of simultaneously detecting and genotyping PPV strains. Several 70-mer oligonucleotide probes were specific for the detection and genotyping of individual PPV isolates to their strains. Other probes were specific for the detection and identification of two or three PPV strains. One probe (universal), derived from the genome highly conserved 3' non-translated region, detected all individual strains of PPV. This universal PPV probe, combined with probes specific for each known strain, could be used for new PPV strain discovery. Finally, indirect fluorescent labeling of cDNA with cyanine after cDNA synthesis enhanced the sensitivity of the virus detection without the use of the PCR amplification step. The PPV microarray detected and identified efficiently the PPV strains in PPV-infected peach, apricot and Nicotiana benthamiana leaves. This PPV detection method is versatile, and enables the simultaneous detection of plant pathogens.

The six Tomato yellow leaf curl virus genes expressed individually in tomato induce different levels of plant stress response attenuation.

Tomato yellow leaf curl virus (TYLCV) is a begomovirus infecting tomato plants worldwide. TYLCV needs a healthy host environment to ensure a successful infection cycle for long periods. Hence, TYLCV restrains its destructive effect and induces neither a hypersensitive response nor cell death in infected tomatoes. On the contrary, TYLCV counteracts cell death induced by other factors, such as inactivation of HSP90 functionality. Suppression of plant death is associated with the inhibition of the ubiquitin 26S proteasome degradation and with a deactivation of the heat shock transcription factor HSFA2 pathways (including decreased HSP17 levels). The goal of the current study was to find if the individual TYLCV genes were capable of suppressing HSP90-dependent death and HSFA2 deactivation. The expression of C2 (C3 and CP to a lesser extent) caused a decrease in the severity of death phenotypes, while the expression of V2 (C1 and C4 to a lesser extent) strengthened cell death. However, C2 or V2 markedly affected stress response under conditions of viral infection. The downregulation of HSFA2 signaling, initiated by the expression of C1 and V2, was detected in the absence of virus infection, but was enhanced in infected plants, while CP and C4 mitigated HSFA2 levels only in the infected tomatoes. The dependence of analyzed plant stress response suppression on the interaction of the expressed genes with the environment created by the whole virus infection was more pronounced than on the expression of individual TYLCV genes.

Tomato yellow leaf curl virus infection mitigates the heat stress response of plants grown at high temperatures.

Cultured tomatoes are often exposed to a combination of extreme heat and infection with Tomato yellow leaf curl virus (TYLCV). This stress combination leads to intense disease symptoms and yield losses. The response of TYLCV-susceptible and resistant tomatoes to heat stress together with viral infection was compared. The plant heat-stress response was undermined in TYLCV infected plants. The decline correlated with the down-regulation of heat shock transcription factors (HSFs) HSFA2 and HSFB1, and consequently, of HSF-regulated genes Hsp17, Apx1, Apx2 and Hsp90. We proposed that the weakened heat stress response was due to the decreased capacity of HSFA2 to translocate into the nuclei of infected cells. All the six TYLCV proteins were able to interact with tomato HSFA2 in vitro, moreover, coat protein developed complexes with HSFA2 in nuclei. Capturing of HSFA2 by viral proteins could suppress the transcriptional activation of heat stress response genes. Application of both heat and TYLCV stresses was accompanied by the development of intracellular large protein aggregates containing TYLCV proteins and DNA. The maintenance of cellular chaperones in the aggregated state, even after recovery from heat stress, prevents the circulation of free soluble chaperones, causing an additional decrease in stress response efficiency.

Diagnosis and control of cereal viruses in the Middle East.

Middle Eastern countries are major consumers of small grain cereals. Egypt is the biggest bread wheat producer with 7.4 million tons (MT) in 2007, but at the same time, it had to import 5.9 MT. Jordan and Israel import almost all the grains they consume. Viruses are the major pathogens that impair grain production in the Middle East, infecting in some years more than 80% of the crop. They are transmitted in nonpersistent, semipersistent, and persistent manners by insects (aphids, leafhoppers, and mites), and through soil and seeds. Hence, cereal viruses have to be controlled, not only in the field but also through the collaborative efforts of the plant quarantine services inland and at the borders, involving all the Middle Eastern countries. Diagnosis of cereal viruses may include symptom observation, immunological technologies such as ELISA using polyclonal and monoclonal antibodies raised against virus coat protein expressed in bacteria, and molecular techniques such as PCR, microarrays, and deep sequencing. In this chapter, we explore the different diagnoses, typing, and detection techniques of cereal viruses available to the Middle Eastern countries. We highlight the plant quarantine service and the prevention methods. Finally, we review the breeding efforts for virus resistance, based on conventional selection and genetic engineering.