The sly-miR166-SlyHB module acts as a susceptibility factor during ToLCNDV infection.

KEY MESSAGE: The role of miRNAs during viral pathogenesis is poorly understood in plants. Here, we demonstrate a miRNA/target module that acts as a susceptibility factor during ToLCNDV infection. Tomato leaf curl New Delhi virus (ToLCNDV) is a devastating pathogen that causes huge crop loss. It is spreading to new geographical locations at a very rapid rate-raising serious concerns. Evolution of insecticidal resistance in Bemisia tabaci which acts as the carrier for ToLCNDV has made insect control very difficult in the recent years. Thus, it is important that the host molecular mechanisms associated with ToLCNDV resistance/susceptibility are investigated to develop management strategies. In our study, we have identified that sly-miR166/SlyHB module acts as a susceptibility factor to ToLCNDV in Solanum lycopersicum. Sly-miR166 is differentially regulated upon ToLCNDV infection in two contrasting tomato cultivars; H-88-78-1 (tolerant to ToLCNDV) and Punjab Chhuhara (susceptible to ToLCNDV). Expression analysis of predicted sly-miR166 targets revealed that the expression of SlyHB is negatively correlated with its corresponding miRNA. Virus-induced gene silencing of SlyHB in the susceptible tomato cultivar resulted in the decrease in disease severity suggesting that SlyHB is a negative regulator of plant defence. In summary, our study highlights a miRNA/target module that acts as a susceptibility factor during ToLCNDV infection. To the best of our knowledge, this is the first report that highlights the role of sly-miR166/SlyHB module in ToLCNDV pathogenesis.

Fungal F8-Culture Filtrate Induces Tomato Resistance against Tomato Yellow Leaf Curl Thailand Virus.

Tomato (Solanum lycopersicum) is an important economic crop worldwide. However, tomato production is jeopardized by the devastating tomato yellow leaf curl disease caused by whitefly-transmitted begomoviruses (WTBs). In this study, we evaluated the efficacy of our previously developed plant antiviral immunity inducer, fungal F8-culture filtrate, on tomato to combat tomato yellow leaf curl Thailand virus (TYLCTHV), the predominant WTB in Taiwan. Our results indicated that F8-culture filtrate treatment induced strong resistance, did not reduce the growth of tomato, and induced prominent resistance against TYLCTHV both in the greenhouse and in the field. Among TYLCTHV-inoculated Yu-Nu tomato grown in the greenhouse, a greater percentage of plants treated with F8-culture filtrate (43-100%) were healthy-looking compared to the H2O control (0-14%). We found that TYLCTHV cannot move systemically only on the F8-culture filtrate pretreated healthy-looking plants. Tracking the expression of phytohormone-mediated immune maker genes revealed that F8-culture filtrate mainly induced salicylic acid-mediated plant immunity. Furthermore, callose depositions and the expression of the pathogen-induced callose synthase gene, POWDERY MILDEW RESISTANT 4 were only strongly induced by TYLCTHV on tomato pretreated with F8-culture filtrate. This study provides an effective way to induce tomato resistance against TYLCTHV.

Defence priming in tomato by the green leaf volatile (Z)-3-hexenol reduces whitefly transmission of a plant virus.

Green leaf volatiles (GLVs) can induce defence priming, that is, can enable plants to respond faster or more strongly to future stress. The effects of priming by GLVs on defence against insect herbivores and pathogens have been investigated, but little is known about the potential of GLVs to prime crops against virus transmission by vector insects. Here, we tested the hypothesis that exposure to the GLV Z-3-hexenol (Z-3-HOL) can prime tomato (Solanum lycopersicum) for an enhanced defence against subsequent Tomato yellow leaf curl virus (TYLCV) transmission by the whitefly Bemisia tabaci. Bioassays showed that Z-3-HOL priming reduced subsequent plant susceptibility to TYLCV transmission by whiteflies. Z-3-HOL treatment increased transcripts of jasmonic acid (JA) biosynthetic genes and increased whitefly-induced transcripts of salicylic acid (SA) biosynthetic genes in plants. Using chemical inducers, transgenics and mutants, we demonstrated that induction of JA reduced whitefly settling and successful whitefly inoculation, while induction of SA reduced TYLCV transmission by whiteflies. Defence gene transcripts and flavonoid levels were enhanced when whiteflies fed on Z-3-HOL-treated plants. Moreover, Z-3-HOL treatment reduced the negative impact of whitefly infestation on tomato growth. These findings suggest that Z-3-HOL priming may be a valuable tool for improving management of insect-transmitted plant viruses.

Development of novel detection system for sweet potato leaf curl virus using recombinant scFv.

Sweet potato leaf curl virus (SPLCV) causes yield losses in sweet potato cultivation. Diagnostic techniques such as serological detection have been developed because these plant viruses are difficult to treat. Serological assays have been used extensively with recombinant antibodies such as whole immunoglobulin or single-chain variable fragments (scFv). An scFv consists of variable heavy (VH) and variable light (VL) chains joined with a short, flexible peptide linker. An scFv can serve as a diagnostic application using various combinations of variable chains. Two SPLCV-specific scFv clones, F7 and G7, were screened by bio-panning process with a yeast cell which expressed coat protein (CP) of SPLCV. The scFv genes were subcloned and expressed in Escherichia coli. The binding affinity and characteristics of the expressed proteins were confirmed by enzyme-linked immunosorbent assay using SPLCV-infected plant leaves. Virus-specific scFv selection by a combination of yeast-surface display and scFv-phage display can be applied to detection of any virus.

Artificially induced phased siRNAs promote virus resistance in transgenic plants.

We previously developed transgenic tobacco plants that were resistant to two geminiviruses. We generated resistance using RNAi constructs that produced trans-acting siRNA (tasiRNA) like secondary siRNAs known as phased siRNA (phasiRNA) that targeted several regions of Tomato Leaf Curl New Delhi Virus (ToLCNDV) and Tomato Leaf Curl Gujarat Virus (ToLCGV) transcripts encoding the RNA silencing suppressor proteins AC2 and AC4. Here, we performed degradome analysis to determine the precise cleavage sites of RNA-RNA interaction between phasiRNA and viral transcripts. We then applied our RNAi technology in tomato, which is the natural host for ToLCNDV and ToLCGV. The relative ease of developing and using phasiRNA constructs represents a significant technical advance in imparting virus resistance in crops and/or important model systems.

Two types of lysozymes from the whitefly Bemisia tabaci: Molecular characterization and functional diversification.

Lysozyme is well-known as an immune effector in the immune system. Here we identified three genes including one c-type lysozyme, Btlysc, and two i-type lysozymes, Btlysi1 and Btlysi2, from the whitefly Bemisia tabaci. All three lysozymes were constitutively expressed in different tissues and developmental stages, but the two types of lysozymes showed different expression patterns. The expression levels of Btlysi1 and Btlysi2 were dramatically induced after the whitefly fed with different host plants while the expression level of Btlysc kept unchanged. After fungal infection and begomovirus acquisition, Btlysc expression was significantly upregulated while Btlysi1 and Btlysi2 expression were basically not induced. Furthermore, we found that Btlysc showed muramidase and antibacterial activities. Altogether, our results suggest that the two types of lysozymes act in two different ways in B. tabaci, that is, Btlysc is involved in the whitefly immune system while Btlysi1 and Btlysi2 may play a role in digestion or nutrition absorption.

Development and application of triple antibody sandwich enzyme-linked immunosorbent assays for begomovirus detection using monoclonal antibodies against Tomato yellow leaf curl Thailand virus.

BACKGROUND: Tomato yellow leaf curl Thailand virus, TYLCTHV, is a begomovirus that causes severe losses of tomato crops in Thailand as well as several countries in Southeast and East Asia. The development of monoclonal antibodies (MAbs) and serological methods for detecting TYLCTHV is essential for epidemiological studies and screening for virus-resistant cultivars. METHODS: The recombinant coat protein (CP) of TYLCTHV was expressed in Escherichia coli and used to generate MAbs against TYLCTHV through hybridoma technology. The MAbs were characterized and optimized to develop triple antibody sandwich enzyme-linked immunosorbent assays (TAS-ELISAs) for begomovirus detection. The efficiency of TAS-ELISAs for begomovirus detection was evaluated with tomato, pepper, eggplant, okra and cucurbit plants collected from several provinces in Thailand. Molecular identification of begomoviruses in these samples was also performed through PCR and DNA sequence analysis of the CP gene. RESULTS: Two MAbs (M1 and D2) were generated and used to develop TAS-ELISAs for begomovirus detection. The results of begomovirus detection in 147 field samples indicated that MAb M1 reacted with 2 begomovirus species, TYLCTHV and Tobacco leaf curl Yunnan virus (TbLCYnV), whereas MAb D2 reacted with 4 begomovirus species, TYLCTHV, TbLCYnV, Tomato leaf curl New Delhi virus (ToLCNDV) and Squash leaf curl China virus (SLCCNV). Phylogenetic analyses of CP amino acid sequences from these begomoviruses revealed that the CP sequences of begomoviruses recognized by the narrow-spectrum MAb M1 were highly conserved, sharing 93% identity with each other but only 72-81% identity with MAb M1-negative begomoviruses. The CP sequences of begomoviruses recognized by the broad-spectrum MAb D2 demonstrated a wider range of amino acid sequence identity, sharing 78-96% identity with each other and 72-91% identity with those that were not detected by MAb D2. CONCLUSIONS: TAS-ELISAs using the narrow-specificity MAb M1 proved highly efficient for the detection of TYLCTHV and TbLCYnV, whereas TAS-ELISAs using the broad-specificity MAb D2 were highly efficient for the detection of TYLCTHV, TbLCYnV, ToLCNDV and SLCCNV. Both newly developed assays allow for sensitive, inexpensive, high-throughput detection of begomoviruses in field plant samples, as well as screening for virus-resistant cultivars.

Mimic Phosphorylation of a ßC1 Protein Encoded by TYLCCNB Impairs Its Functions as a Viral Suppressor of RNA Silencing and a Symptom Determinant.

Phosphorylation of the ßC1 protein encoded by the betasatellite of tomato yellow leaf curl China virus (TYLCCNB-ßC1) by SNF1-related protein kinase 1 (SnRK1) plays a critical role in defense of host plants against geminivirus infection in Nicotiana benthamiana However, how phosphorylation of TYLCCNB-ßC1 impacts its pathogenic functions during viral infection remains elusive. In this study, we identified two additional tyrosine residues in TYLCCNB-ßC1 that are phosphorylated by SnRK1. The effects of TYLCCNB-ßC1 phosphorylation on its functions as a viral suppressor of RNA silencing (VSR) and a symptom determinant were investigated via phosphorylation mimic mutants in N. benthamiana plants. Mutations that mimic phosphorylation of TYLCCNB-ßC1 at tyrosine 5 and tyrosine 110 attenuated disease symptoms during viral infection. The phosphorylation mimics weakened the ability of TYLCCNB-ßC1 to reverse transcriptional gene silencing and to suppress posttranscriptional gene silencing and abolished its interaction with N. benthamiana ASYMMETRIC LEAVES 1 in N. benthamiana leaves. The mimic phosphorylation of TYLCCNB-ßC1 had no impact on its protein stability, subcellular localization, or self-association. Our data establish an inhibitory effect of phosphorylation of TYLCCNB-ßC1 on its pathogenic functions as a VSR and a symptom determinant and provide a mechanistic explanation of how SnRK1 functions as a host defense factor.IMPORTANCE Tomato yellow leaf curl China virus (TYLCCNV), which causes a severe yellow leaf curl disease in China, is a monopartite geminivirus associated with the betasatellite (TYLCCNB). TYLCCNB encodes a single pathogenicity protein, ßC1 (TYLCCNB-ßC1), which functions as both a viral suppressor of RNA silencing (VSR) and a symptom determinant. Here, we show that mimicking phosphorylation of TYLCCNB-ßC1 weakens its ability to reverse transcriptional gene silencing, to suppress posttranscriptional gene silencing, and to interact with N. benthamiana ASYMMETRIC LEAVES 1. To our knowledge, this is the first report establishing an inhibitory effect of phosphorylation of TYLCCNB-ßC1 on its pathogenic functions as both a VSR and a symptom determinant and to provide a mechanistic explanation of how SNF1-related protein kinase 1 acts as a host defense factor. These findings expand the scope of phosphorylation-mediated defense mechanisms and contribute to further understanding of plant defense mechanisms against geminiviruses.

A rapid virus-induced gene silencing (VIGS) method for assessing resistance and susceptibility to cassava mosaic disease.

BACKGROUND: Cassava mosaic disease (CMD) is a major constraint to cassava production in sub-Saharan Africa. Under field conditions, evaluation for resistance to CMD takes 12-18 months, often conducted across multiple years and locations under pressure from whitefly-mediated transmission. Under greenhouse or laboratory settings, evaluation for resistance or susceptibility to CMD involves transmission of the causal viruses from an infected source to healthy plants through grafting, or by using Agrobacterium-mediated or biolistic delivery of infectious clones. Following inoculation, visual assessment for CMD symptom development and recovery requires 12-22 weeks. Here we report a rapid screening system for determining resistance and susceptibility to CMD based on virus-induced gene silencing (VIGS) of an endogenous cassava gene. RESULTS: A VIGS vector was developed based on an infectious clone of the virulent strain of East African cassava mosaic virus (EACMV-K201). A sequence from the cassava (Manihot esculenta) ortholog of Arabidopsis SPINDLY (SPY) was cloned into the CP position of the DNA-A genomic component and used to inoculate cassava plants by Helios® Gene Gun microparticle bombardment. Silencing of Manihot esculenta SPY (MeSPY) using MeSPY1-VIGS resulted in shoot-tip necrosis followed by death of the whole plant in CMD susceptible cassava plants within 2-4 weeks. CMD resistant cultivars were not affected and remained healthy after challenge with MeSPY1-VIGS. Significantly higher virus titers were detected in CMD-susceptible cassava lines compared to resistant controls and were correlated with a concomitant reduction in MeSPY expression in susceptible plants. CONCLUSIONS: A rapid VIGS-based screening system was developed for assessing resistance and susceptibility to CMD. The method is space and resource efficient, reducing the time required to perform CMD screening to as little as 2-4 weeks. It can be employed as a high throughput rapid screening system to assess new cassava cultivars and for screening transgenic, gene edited and breeding lines under controlled growth conditions.

The Whitefly Bemisia tabaci Knottin-1 Gene Is Implicated in Regulating the Quantity of Tomato Yellow Leaf Curl Virus Ingested and Transmitted by the Insect.

The whitefly Bemisia tabaci is a major pest to agricultural crops. It transmits begomoviruses, such as Tomato yellow leaf curl virus (TYLCV), in a circular, persistent fashion. Transcriptome analyses revealed that B. tabaci knottin genes were responsive to various stresses. Upon ingestion of tomato begomoviruses, two of the four knottin genes were upregulated, knot-1 (with the highest expression) and knot-3. In this study, we examined the involvement of B. tabaci knottin genes in relation to TYLCV circulative transmission. Knottins were silenced by feeding whiteflies with knottin dsRNA via detached tomato leaves. Large amounts of knot-1 transcripts were present in the abdomen of whiteflies, an obligatory transit site of begomoviruses in their circulative transmission pathway; knot-1 silencing significantly depleted the abdomen from knot-1 transcripts. Knot-1 silencing led to an increase in the amounts of TYLCV ingested by the insects and transmitted to tomato test plants by several orders of magnitude. This effect was not observed following knot-3 silencing. Hence, knot-1 plays a role in restricting the quantity of virions an insect may acquire and transmit. We suggest that knot-1 protects B. tabaci against deleterious effects caused by TYLCV by limiting the amount of virus associated with the whitefly vector.

Tomato yellow leaf curl virus confronts host degradation by sheltering in small/midsized protein aggregates.

Tomato yellow leaf curl virus (TYLCV) is a begomovirus transmitted by the whitefly Bemisia tabaci to tomato and other crops. TYLCV proteins are endangered by the host defenses. We have analyzed the capacity of the tomato plant and of the whitefly insect vector to degrade the six proteins encoded by the TYLCV genome. Tomato and whitefly demonstrated the highest proteolytic activity in the fractions containing soluble proteins, less-in large protein aggregates; a significant decrease of TYLCV proteolysis was detected in the intermediate-sized aggregates. All the six TYLCV proteins were differently targeted by the cytoplasmic and nuclear degradation machineries (proteases, ubiquitin 26S proteasome, autophagy). TYLCV could confront host degradation by sheltering in small/midsized aggregates, where viral proteins are less exposed to proteolysis. Indeed, TYLCV proteins were localized in aggregates of various sizes in both host organisms. This is the first study comparing degradation machinery in plant and insect hosts targeting all TYLCV proteins.

NIK1, a host factor specialized in antiviral defense or a novel general regulator of plant immunity?

NIK1 is a receptor-like kinase involved in plant antiviral immunity. Although NIK1 is structurally similar to the plant immune factor BAK1, which is a key regulator in plant immunity to bacterial pathogens, the NIK1-mediated defenses do not resemble BAK1 signaling cascades. The underlying mechanism for NIK1 antiviral immunity has recently been uncovered. NIK1 activation mediates the translocation of RPL10 to the nucleus, where it interacts with LIMYB to fully down-regulate translational machinery genes, resulting in translation inhibition of host and viral mRNAs and enhanced tolerance to begomovirus. Therefore, the NIK1 antiviral immunity response culminates in global translation suppression, which represents a new paradigm for plant antiviral defenses. Interestingly, transcriptomic analyses in nik1 mutant suggest that NIK1 may suppress antibacterial immune responses, indicating a possible opposite effect of NIK1 in bacterial and viral infections.

Artificial TALE as a Convenient Protein Platform for Engineering Broad-Spectrum Resistance to Begomoviruses.

Transcription activator-like effectors (TALEs) are a class of sequence-specific DNA-binding proteins that utilize a simple and predictable modality to recognize target DNA. This unique characteristic allows for the rapid assembly of artificial TALEs, with high DNA binding specificity, to any target DNA sequences for the creation of customizable sequence-specific nucleases used in genome engineering. Here, we report the use of an artificial TALE protein as a convenient platform for designing broad-spectrum resistance to begomoviruses, one of the most destructive plant virus groups, which cause tremendous losses worldwide. We showed that artificial TALEs, which were assembled based on conserved sequence motifs within begomovirus genomes, could confer partial resistance in transgenic Nicotiana benthamiana to all three begomoviruses tested. Furthermore, the resistance was maintained even in the presence of their betasatellite. These results shed new light on the development of broad-spectrum resistance against DNA viruses, such as begomoviruses.

Recessive Resistance Derived from Tomato cv. Tyking-Limits Drastically the Spread of Tomato Yellow Leaf Curl Virus.

The tomato yellow leaf curl disease (TYLCD) causes severe damage to tomato (Solanum lycopersicum L.) crops throughout tropical and subtropical regions of the world. TYLCD is associated with a complex of single-stranded circular DNA plant viruses of the genus Begomovirus (family Geminiviridae) transmitted by the whitefy Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae). The tomato inbred line TX 468-RG is a source of monogenic recessive resistance to begomoviruses derived from the hybrid cv. Tyking F1. A detailed analysis of this germplasm source against tomato yellow leaf curl virus-Israel (TYLCV-IL), a widespread TYLCD-associated virus, showed a significant restriction to systemic virus accumulation even under continuous virus supply. The resistance was effective in limiting the onset of TYLCV-IL in tomato, as significantly lower primary spread of the virus occurred in resistant plants. Also, even if a limited number of resistant plants could result infected, they were less efficient virus sources for secondary spread owing to the impaired TYLCV-IL accumulation. Therefore, the incorporation of this resistance into breeding programs might help TYLCD management by drastically limiting TYLCV-IL spread.

Lost in translation: an antiviral plant defense mechanism revealed.

Plant viruses depend on the host translational machinery to establish their infectious cycle. In a recent Nature publication, Zorzatto et al. (2015) highlight the suppression of the protein synthesis process as an antiviral defense mechanism in plants.

Chilli leaf curl virus infection highlights the differential expression of genes involved in protein homeostasis and defense in resistant chilli plants.

Geminiviruses have evolved with tremendous potential of recombination and possess the ability to manipulate several cellular processes of hosts. Chilli leaf curl virus (ChiLCV) is a monopartite Begomovirus (family Geminiviridae) which has emerged as a serious threat to chilli production worldwide. To date, development of resistant chilli varieties through conventional plant breeding techniques remains the major antiviral strategy. To explore the potential resistance factors in Capsicum annuum var. Punjab Lal, we performed a transcriptome analysis in ChiLCV-infected plants by exploiting the advantage of sensitivity and efficiency of suppression subtractive hybridization (SSH). Out of 480 clones screened, 231 unique expressed sequence tags (ESTs) involved in different cellular and physiological processes were identified. An interactome network of ChiLCV responsive differentially expressed genes revealed an array of proteins involved in key cellular processes including transcription, replication, photosynthesis, and defense. A comparative study of gene expression between resistant and susceptible chilli plants revealed upregulation of several defense-related genes such as nucleotide-binding site leucine-rich repeat (NBS-LRR) domain containing protein, lipid transfer protein, thionin, polyphenol oxidase, and other proteins like ATP/ADP transporter in the ChiLCV-resistant variety. Taken together, the present study provides novel insights into the transcriptomics of ChiLCV-resistant chilli plants.

NIK1-mediated translation suppression functions as a plant antiviral immunity mechanism.

Plants and plant pathogens are subject to continuous co-evolutionary pressure for dominance, and the outcomes of these interactions can substantially impact agriculture and food security. In virus-plant interactions, one of the major mechanisms for plant antiviral immunity relies on RNA silencing, which is often suppressed by co-evolving virus suppressors, thus enhancing viral pathogenicity in susceptible hosts. In addition, plants use the nucleotide-binding and leucine-rich repeat (NB-LRR) domain-containing resistance proteins, which recognize viral effectors to activate effector-triggered immunity in a defence mechanism similar to that employed in non-viral infections. Unlike most eukaryotic organisms, plants are not known to activate mechanisms of host global translation suppression to fight viruses. Here we demonstrate in Arabidopsis that the constitutive activation of NIK1, a leucine-rich repeat receptor-like kinase (LRR-RLK) identified as a virulence target of the begomovirus nuclear shuttle protein (NSP), leads to global translation suppression and translocation of the downstream component RPL10 to the nucleus, where it interacts with a newly identified MYB-like protein, L10-INTERACTING MYB DOMAIN-CONTAINING PROTEIN (LIMYB), to downregulate translational machinery genes fully. LIMYB overexpression represses ribosomal protein genes at the transcriptional level, resulting in protein synthesis inhibition, decreased viral messenger RNA association with polysome fractions and enhanced tolerance to begomovirus. By contrast, the loss of LIMYB function releases the repression of translation-related genes and increases susceptibility to virus infection. Therefore, LIMYB links immune receptor LRR-RLK activation to global translation suppression as an antiviral immunity strategy in plants.

RNAi mediated gene silencing against betasatellite associated with Croton yellow vein mosaic begomovirus.

Plant viruses encode suppressors of posttranscriptional gene silencing, an adaptive antiviral defense responses that confines virus infection. Previously, we identified single-stranded DNA satellite (also known as DNA-ß) of ~1,350 nucleotides in length associated with Croton yellow vein mosaic begomovirus (CYVMV) in croton plants. The expression of genes from DNA-ß requires the begomovirus for packaged, replication, insect transmission and movement in plants. The present study demonstrates the effect of the ßC1 gene on the silencing pathway as analysed by using both transgenic systems and transient Agrobacterium tumefaciens based delivery. Plants that carry an intron-hairpin construct covering the ßC1 gene accumulated cognate small-interfering RNAs and remained symptom-free after exposure to CYVMV and its satellite. These results suggest that ßC1 interferes with silencing mechanism.

Degradation mechanisms of the Tomato yellow leaf curl virus coat protein following inoculation of tomato plants by the whitefly Bemisia tabaci.

BACKGROUND: Tomato yellow leaf curl virus (TYLCV) is a begomovirus infecting tomato cultures worldwide. TYLCV is transmitted to plants by the whitefly Bemisia tabaci. Once in the plant, the virus is subjected to attack by the host-plant defences, which may include sequestration in aggregates, proteolysis, ubiquitination, 26S proteasome degradation and autophagy. Elucidating how the virus avoids destruction will make it possible to understand infection and possibly devise countermeasures. RESULTS: The accumulation of viral coat protein (CP) and of viral DNA in plants is a marker of a successful virus transmission by B. tabaci. In response to infection, tomato tissues display multiple ways of degrading TYLCV proteins and DNA. In this study it is shown that CP (in soluble and insoluble states) is the target of protease digestion, 26S proteasome degradation and autophagy. The highest degradation capacity was detected among soluble proteins and proteins in large aggregates/inclusion bodies; cytoplasmic extracts displayed higher activity than nuclear fractions. The very same fractions possessed the highest capacity to degrade viral genomic DNA. Separately, 26S proteasome degradation was associated with large aggregates (more pronounced in the nuclear than in the cytoplasmic fractions), which are indicators of a successful abduction of plants by viruses. Autophagy/lysosome/vacuole degradation was a characteristic of intermediate aggregates, sequestering the CP in the cytoplasm and retarding the development of large aggregates. Chloroplast proteases were active in soluble as well as in insoluble protein extracts. CONCLUSIONS: To the best of the authors' knowledge, this study is the first attempt to identify elements of the virus-targeted degradation machinery, which is a part of the plant response to virus invasion.

Eugenol enhances the resistance of tomato against tomato yellow leaf curl virus.

BACKGROUND: Tomato yellow leaf curl virus disease (TYLCVD) causes severe to economic losses in tomato crops in China. The control of TYLCVD is based primarily on the use of synthetic insecticide to control its vector whitefly (Bemisia tabaci). To look for an alternative method for disease control, we investigated the effect of eugenol on controlling TYLCVD. The potential of eugenol to trigger systemic acquired resistance (SAR) in tomato (Jiangsu 14) plants against TYLCV was also investigated. RESULTS: In greenhouse experiments, eugenol significantly reduced disease severity when applied as a foliar spray, thus demonstrating a systemic effect. The disease spread rapidly in control plants and by the end of the experiment almost all control plants showed severe symptoms. Eugenol also induced H2O2 accumulation in tomato plants. Activities of peroxidase (POD), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) were significantly induced compared with those of control plants. As further consequences, increase of salicylic acid (SA) levels and expression of PR-1 proteins, a molecular marker of SAR in tomato, could also be observed. CONCLUSION: This is the first report of eugenol as an elicitor and its ability to suppress plant virus diseases under greenhouse conditions. It is suggested that eugenol has the potential to be an effective biocontrol agent against TYLCV in tomato plants.