Systemic Symptom Development Research Articles

Complementary roles of EPS, T3SS and Expansin for virulence of Erwinia tracheiphila, the causative agent of cucurbit wilt.

Erwinia tracheiphila (Smith) is a recently emerged plant pathogen that causes severe economic losses in cucurbit crops in temperate Eastern North America. E. tracheiphila is xylem restricted, and virulence is thought to be related to Exopolysaccharides (EPS) and biofilm formation, which occlude the passage of sap in xylem vessels and causes systemic wilt. However, the role of EPS and biofilm formation, and their contribution to disease in relation to other virulence loci are unknown. Here, we use deletion mutants to explore the roles of EPS, Hrp Type III secretion system (Hrp T3SS) and Expansin in plant colonization and virulence. Then, we quantify the expression of the genes encoding these factors during infection. Our results show that Exopolysaccharides are essential for E. tracheiphila survival in host plants, while Hrp T3SS and Expansin are dispensable for survival but needed for systemic wilt symptom development. EPS and Hrp T3SS display contrasting expression patterns in the plant, reflecting their relevance in different stages of the infection. Finally, we show that expression of the eps and hrpT3SS operons is downregulated in mildly increased temperatures, suggesting a link between expression of these virulence factors and geographic restriction of E. tracheiphila to temperate regions. Our work highlights how E. tracheiphila virulence is a complex trait where several loci are coordinated during infection. These results further shed light into the relationship between virulence factors and the ecology of this pathosystem, which will be essential for developing sustainable management strategies for this emerging pathogen.

Synergy between an emerging monopartite begomovirus and a DNA-B component

In recent decades, a legion of monopartite begomoviruses transmitted by the whitefly Bemisia tabaci has emerged as serious threats to vegetable crops in Africa. Recent studies in Burkina Faso (West Africa) reported the predominance of pepper yellow vein Mali virus (PepYVMLV) and its frequent association with a previously unknown DNA-B component. To understand the role of this DNA-B component in the emergence of PepYVMLV, we assessed biological traits related to virulence, virus accumulation, location in the tissue and transmission. We demonstrate that the DNA-B component is not required for systemic movement and symptom development of PepYVMLV (non-strict association), but that its association produces more severe symptoms including growth arrest and plant death. The increased virulence is associated with a higher viral DNA accumulation in plant tissues, an increase in the number of contaminated nuclei of the phloem parenchyma and in the transmission rate by B. tabaci. Our results suggest that the association of a DNA-B component with the otherwise monopartite PepYVMLV is a key factor of its emergence.

Systemic infection and symptom development of agro-inoculated cDNA clone of cherry rusty mottle-associated virus in sweet cherry (Prunus avium)

Cherry rusty mottle-associated virus (CRMaV), which belongs the genus Robigovirus of the family Betaflexiviridae, is strongly associated with cherry rusty mottle disease of sweet cherry, Prunus avium. Here, we report on the successful development of an Agrobacterium-based inoculation system for a cloned CRMaV cDNA construct. Agro-inoculation of virus-free cherry rootstock ‘Krymsk6’ [P. cerasus x (P. cerasus x P. maackii)] resulted in the development of chlorotic yellow mottle symptoms on systemic leaves beginning at 50 days post inoculation. The presence of CRMaV in ‘Krymsk6’ agro-inoculated plants was confirmed by RT-PCR and ELISA. Subsequently, CRMaV from agro-inoculated ‘Krymsk6’ was graft-transmissible onto virus-free sweet cherry rootstock P. avium ‘Mazzard’ as evidenced by the production of typical cherry rusty mottle symptoms beginning at 35 days post grafting, and further confirmed by western blotting and RT-PCR. These results showed conclusively that CRMaV is the causal agent of cherry rusty mottle disease in sweet cherry. The reverse genetic system presented in this study can be used as a tool to investigate the molecular biology of CRMaV and also a template for infectious clone development for other viruses in the genus Robigovirus.

Xylella fastidiosa subsp. pauca Strains Fb7 and 9a5c from Citrus Display Differential Behavior, Secretome, and Plant Virulence.

Xylella fastidiosa colonizes the xylem of various cultivated and native plants worldwide. Citrus production in Brazil has been seriously affected, and major commercial varieties remain susceptible to Citrus Variegated Chlorosis (CVC). Collective cellular behaviors such as biofilm formation influence virulence and insect transmission of X. fastidiosa. The reference strain 9a5c produces a robust biofilm compared to Fb7 that remains mostly planktonic, and both were isolated from symptomatic citrus trees. This work deepens our understanding of these distinct behaviors at the molecular level, by comparing the cellular and secreted proteomes of these two CVC strains. Out of 1017 identified proteins, 128 showed differential abundance between the two strains. Different protein families were represented such as proteases, hemolysin-like proteins, and lipase/esterases, among others. Here we show that the lipase/esterase LesA is among the most abundant secreted proteins of CVC strains as well, and demonstrate its functionality by complementary activity assays. More severe symptoms were observed in Nicotiana tabacum inoculated with strain Fb7 compared to 9a5c. Our results support that systemic symptom development can be accelerated by strains that invest less in biofilm formation and more in plant colonization. This has potential application in modulating the bacterial-plant interaction and reducing disease severity.

Wheat root transcriptional responses against Gaeumannomyces graminis var. tritici

Wheat root rot caused by Gaeumannomyces graminis var. tritici (Ggt) results in severe yield losses in wheat production worldwide. However, little is known about the molecular mechanism that regulates systemic symptom development in infected wheat. Fluorescent microscopy observation of the stained wheat roots infected by Ggt showed that lesions were visible when the fungus could be detected in the endodermis, pericycle and phloem at 5 days post inoculation (dpi), and rust symptoms were visible when there was extensive fungal colonization in the root cortex at 6 dpi. Transcriptome sequencing of Ggt-inoculated wheat roots and healthy control root samples was performed at 5 dpi to identify Ggt-induced gene expression changes in wheat roots at the time of lesion formation. A total of 3973 differentially expressed genes (DEGs) were identified, of which 1004 (25.27%) were up-regulated and 2969 (74.73%) were down-regulated in Ggt-inoculated wheat roots compared with those in control roots. GO annotation and KEGG pathway analysis of these DEGs revealed that many of them were associated with pathogen resistance, such as those involved in oxidation-reduction process, tryptophan biosynthesis process, and phenylpropanoid biosynthesis process. Analysis of DEGs revealed that 15 DEGs were involved in cellular regulation, 57 DEGs in signal transduction pathways, and 75 DEGs in cell wall reorganization, and 23 DEGs are pathogenesis-related proteins. Reverse transcription quantitative PCR (RT-qPCR) of 13 of those DEGs showed that these genes may play roles in wheat resistance against Ggt. Overall, this study represents the first transcriptional profiling of wheat roots in response to Ggt infection and further characterization of DEGs identified in this study may lead to better understanding of resistance against take-all in wheat.

Genome-Wide Transcriptomic Analysis Reveals Insights into the Response to Citrus bark cracking viroid (CBCVd) in Hop (Humulus lupulus L.).

Viroids are smallest known pathogen that consist of non-capsidated, single-stranded non-coding RNA replicons and they exploits host factors for their replication and propagation. The severe stunting disease caused by Citrus bark cracking viroid (CBCVd) is a serious threat, which spreads rapidly within hop gardens. In this study, we employed comprehensive transcriptome analyses to dissect host-viroid interactions and identify gene expression changes that are associated with disease development in hop. Our analysis revealed that CBCVd-infection resulted in the massive modulation of activity of over 2000 genes. Expression of genes associated with plant immune responses (protein kinase and mitogen-activated protein kinase), hypersensitive responses, phytohormone signaling pathways, photosynthesis, pigment metabolism, protein metabolism, sugar metabolism, and modification, and others were altered, which could be attributed to systemic symptom development upon CBCVd-infection in hop. In addition, genes encoding RNA-dependent RNA polymerase, pathogenesis-related protein, chitinase, as well as those related to basal defense responses were up-regulated. The expression levels of several genes identified from RNA sequencing analysis were confirmed by qRT-PCR. Our systematic comprehensive CBCVd-responsive transcriptome analysis provides a better understanding and insights into complex viroid-hop plant interaction. This information will assist further in the development of future measures for the prevention of CBCVd spread in hop fields.

Tomato Spotted Wilt Virus NSs Protein Supports Infection and Systemic Movement of a Potyvirus and Is a Symptom Determinant.

Plant viruses are inducers and targets of antiviral RNA silencing. To condition susceptibility, most plant viruses encode silencing suppressor proteins that interfere with antiviral RNA silencing. The NSs protein is an RNA silencing suppressor in orthotospoviruses, such as the tomato spotted wilt virus (TSWV). The mechanism of RNA silencing suppression by NSs and its role in virus infection and movement are poorly understood. Here, we cloned and tagged TSWV NSs and expressed it from a GFP-tagged turnip mosaic virus (TuMV-GFP) carrying either a wild-type or suppressor-deficient (AS9) helper component proteinase (HC-Pro). When expressed in cis, NSs restored pathogenicity and promoted systemic infection of suppressor-deficient TuMV-AS9-GFP in Nicotiana benthamiana and Arabidopsis thaliana. Inactivating mutations were introduced in NSs RNA-binding domain one. A genetic analysis with active and suppressor-deficient NSs, in combination with wild-type and mutant plants lacking essential components of the RNA silencing machinery, showed that the NSs insert is stable when expressed from a potyvirus. NSs can functionally replace potyviral HC-Pro, condition virus susceptibility, and promote systemic infection and symptom development by suppressing antiviral RNA silencing through a mechanism that partially overlaps that of potyviral HC-Pro. The results presented provide new insight into the mechanism of silencing suppression by NSs and its effect on virus infection.

Transcriptome analysis of maize leaf systemic symptom infected by Bipolaris zeicola.

Bipolaris zeicola is a fungal pathogen that causes Northern corn leaf spot (NCLS), which is a serious foliar disease in maize and one of the most significant pathogens affecting global food security. Here, we report a genome-wide transcriptional profile analysis using next-generation sequencing (NGS) of maize leaf development after inoculation with B. zeicola. We performed High-Throughput Digital Gene Expression analysis to identify differentially expressed genes (DEGs) in resistant inbred Mo17 lines after infection with B. zeicola at four successive disease development stages—CP (contact period), PP (penetration period), IP (incubation period), and DP (disease period); the expression of the genes was compared with those in a CK (mock-treatment) control. In addition, a sensitive maize line (Zheng58) was used for the comparisons with the Mo17. Among all tested genes, 466 differentially expressed genes were identified in all libraries, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of these genes suggested that they are involved in many biological processes related to systemic symptom development, such as plant hormone signal transduction, starch and sucrose metabolism, phenylpropanoid biosynthesis and photosynthesis. Our systematic analysis provides comprehensive transcriptomic information regarding systemic symptom development in fungal-infected plants. This information will help in furthering our understanding of the detailed mechanisms of plant responses to fungal infection.

Transcriptome analysis of Nicotiana tabacum infected by Cucumber mosaic virus during systemic symptom development.

Virus infection of plants may induce a variety of disease symptoms. However, little is known about the molecular mechanism of systemic symptom development in infected plants. Here we performed the first next-generation sequencing study to identify gene expression changes associated with disease development in tobacco plants (Nicotiana tabacum cv. Xanthi nc) induced by infection with the M strain of Cucumber mosaic virus (M-CMV). Analysis of the tobacco transcriptome by RNA-Seq identified 95,916 unigenes, 34,408 of which were new transcripts by database searches. Deep sequencing was subsequently used to compare the digital gene expression (DGE) profiles of the healthy plants with the infected plants at six sequential disease development stages, including vein clearing, mosaic, severe chlorosis, partial and complete recovery, and secondary mosaic. Thousands of differentially expressed genes were identified, and KEGG pathway analysis of these genes suggested that many biological processes, such as photosynthesis, pigment metabolism and plant-pathogen interaction, were involved in systemic symptom development. Our systematic analysis provides comprehensive transcriptomic information regarding systemic symptom development in virus-infected plants. This information will help further our understanding of the detailed mechanisms of plant responses to viral infection.

Analysis of Symptom Determinant of Cucumber mosaic virus RNA3 via Pseudorecombinant Virus in Zucchini Squash

Isolates of Cucumber mosaic virus (CMV) collected in Korea, were compared with their pathological features in tobacco and zucchini squash. Full-length cDNA clone of RNA3 was generated by using long-distance RT-PCR. Transcript RNA3 from the cDNA clone was inoculated onto host plants with transcripts RNA1 and RNA2 of Fny strain, generating RNA3-pseudorecombinant CMV. Timing and severity of systemic symptom was not significantly different among the pseudorecombinant CMVs in tobacco, compared with strains Fny-CMV and Pf-CMV. However, the pseudorecombinant CMVs induced two different systemic symptoms (mosaic vs. chlorotic spot) in zucchini squash. Based on symptom induction, the pseudorecombinant CMVs were categorized into two classes. The severity and timing of symptoms were correlated with viral RNA accumulations in systemic leaves of zucchini squash, suggesting that different kinetics of virus movement associated with CMV proteins are crucial for systemic infection and symptom development in zucchini squash. The analysis of movement proteins (MP) of CMV strains showed high sequence homology, but the differences of several amino acids were found in the C-terminal region between Class-I-CMV and Class-II-CMV. The analysis of coat proteins (CP) showed that the CMV isolates tested belonged to CMV subgroup I and the viruses shared overall 87-99% sequence identity in their genomes. Phylogenetic analysis of MP and CP suggested that biological properties of Korean CMV isolates have relationships associated with host species.

Modeling-based characterization of the elicitor function of amino acid 461 of Cucumber mosaic virus 1a protein in the hypersensitive response

The Ns strain of Cucumber mosaic virus (CMV) induces hypersensitive response (HR) on Nicotiana tabacum cv. Xanthi-nc and on Nicotiana glutinosa. The genetic determinant of the HR induction was localized earlier to amino acid 461 of the 1a protein. The 3D structure of the 1a protein is still unknown and building a homology model is impossible. Nevertheless, on the basis of secondary structure predictions we have created partial protein models for the region surrounding residue 461 which can account structurally for the effect of aa 461 on elicitor function. Seven different amino acid mutations were designed and introduced to the position 461 of the 1a protein in RNA 1. Three of the mutations (proline, glutamic acid, asparagine) inhibited virus replication. Two of the mutants caused systemic symptom development (lysine and arginine). Two mutants (alanine and serine) resulted in localization of the virus, but strong necrosis similar to the original Ns-CMV strain was not observed. Inoculation of purified Ns-CMV virions at extremely high concentration provoked systemic symptoms.

Cucumber mosaic virus 2a polymerase and 3a movement proteins independently affect both virus movement and the timing of symptom development in zucchini squash

The basis for differences in the timing of systemic symptom elicitation in zucchini squash between a pepper strain of Cucumber mosaic virus (Pf-CMV) and a cucurbit strain (Fny-CMV) was analysed. The difference in timing of appearance of systemic symptoms was shown to map to both RNA 2 and RNA 3 of Pf-CMV, with pseudorecombinant viruses containing either RNA 2 or RNA 3 from Pf-CMV showing an intermediate rate of systemic symptom development compared with those containing both or neither Pf-CMV RNAs. Symptom phenotype was shown to map to two single-nucleotide changes, both in codons for Ile at aa 267 and 168 (in Fny-CMV RNAs 2 and 3, respectively) to Thr (in Pf-CMV RNAs 2 and 3). The differential rate of symptom development was shown to be due to differences in the rates of cell-to-cell movement in the inoculated cotyledons, as well as differences in the rate of egress of the virus from the inoculated leaves. These data indicate that both the CMV 3a movement protein and the CMV 2a polymerase protein affect the rate of movement of CMV in zucchini squash and that these two proteins function independently of each other in their interactions with the host, facilitating virus movement.

Movement of Zucchini yellow mosaic vims Involved in Symptom Severity on Zucchini Squash

Zucchini squash (Cucurbita pepo cv. Black Beauty) plants infected with A strain of Zucchini yellow mosaic virus (ZYMV-A) isolated from a hollyhock plant showed systemically severe mosaic symptom, similar to previously established Cu strain of ZYMV. However, initial symptom of squash infected by ZYMV-A strain was generally more severe than those infected by ZYMV-Cu. Using leaf-detachment assay, examination of kinetics of accumulation of the coat protein (CP) in systemic loaves of squash plants showed that CPs of ZYMV-A appeared earlier than those of ZYMV-Cu. However, both ZYMV-A and ZYMV-Cu showed similar kinetics of CP accumulation 7 days post-inoculation. These results indicate that different rates and initial severity of systemic symptom development were due to differences in the rate of movement rather than vims replication.

Production of Bean yellow mosaic virus resistant subterranean clover (Trifolium subterraneum) plants by transformation with the virus coat protein gene

SUMMARYTransgenic lines of subterranean clover were constructed that contained three different Bean yellow mosaic virus (BYMV) coat protein (CP) gene constructs; full‐length CP, the core region of the CP, and full‐length CP plus the 3′ untranslated region of the viral genome. Transgenic plants containing the full‐length and core CP gene constructs showed high and moderate levels of BYMV resistance. Resistance was measured as a lack or amelioration of viral disease symptoms, which was correlated with a reduction in virus levels and yield loss. A range of different resistance phenotypes was observed. They included reduced infection rates, delay and reduction in local lesion development, and delay and reduction in severity of systemic symptom development. Resistance levels were not correlated with transgene mRNA levels and no transgene‐encoded protein was detected in any of the transgenic lines. This is the first example of genetically engineered virus resistance in a clover.

A phage single-stranded DNA (ssDNA) binding protein complements ssDNA accumulation of a geminivirus and interferes with viral movement.

Geminiviruses are plant viruses with circular single-stranded DNA (ssDNA) genomes encapsidated in double icosahedral particles. Tomato leaf curl geminivirus (ToLCV) requires coat protein (CP) for the accumulation of ssDNA in protoplasts and in plants but not for systemic infection and symptom development in plants. In the absence of CP, infected protoplasts accumulate reduced levels of ssDNA and increased amounts of double-stranded DNA (dsDNA), compared to accumulation in the presence of wild-type virus. To determine whether the gene 5 protein (g5p), a ssDNA binding protein from Escherichia coli phage M13, could restore the accumulation of ssDNA, ToLCV that lacked the CP gene was modified to express g5p or g5p fused to the N-terminal 66 amino acids of CP (CP66:6G:g5). The modified viruses led to the accumulation of wild-type levels of ssDNA and high levels of dsDNA. The accumulation of ssDNA was apparently due to stable binding of g5p to viral ssDNA. The high levels of dsDNA accumulation during infections with the modified viruses suggested a direct role for CP in viral DNA replication. ToLCV that produced the CP66:6G:g5 protein did not spread efficiently in Nicotiana benthamiana plants, and inoculated plants developed only very mild symptoms. In infected protoplasts, the CP66:6G:g5 protein was immunolocalized to nuclei. We propose that the fusion protein interferes with the function of the BV1 movement protein and thereby prevents spread of the infection.

Transgenic tobacco plants expressing the coat protein of cucumber mosaic virus show different virus resistance

Transgenic tobacco (Nicotiana tabacum cv. Xanthi-nc) plants were regenerated after cocultivation of leaf explants withAgrobacterium tumefaciens strain LBA4404 harboring a plasmid that contained the coat protein (CP) gene of cucumber mosaic virus (CMV-As). PCR and Southern blot analyses revealed that the CMV CP gene was successfully introduced into the genomic DNA of the transgenic tobacco plants. Transgenic plants (CP+) expressing CP were obtained and used for screening the virus resistance. They could be categorized into three types after inoculation with the virus: virus-resistant, delay of symptom development, and susceptible type. Most of the CP+ transgenic tobacco plants failed to develop symptoms or showed systemic symptom development delayed for 5 to 42 days as compared to those of nontransgenic control plants after challenged with the same virus. However, some CP+ transgenic plants were highly susceptible after inoculation with the virus. Our results suggest that the CP-mediated viral resistance is readily applicable to CMV disease in other crops.

The 126- and 183-kilodalton proteins of tobacco mosaic virus, and not their common nucleotide sequence, control mosaic symptom formation in tobacco.

Nucleotide substitutions at two positions within the open reading frame encoding the 126-kDa protein in the attenuated masked (M) strain of tobacco mosaic tobamovirus (TMV) to those found in the virulent U1-TMV genome led to the induction of near U1-TMV-like symptoms on leaves of Nicotiana tabacum L. cv. Xanthi nn by progeny virus (M. H. Shintaku, S. A. Carter, Y. Bao, and R. S. Nelson, Virology 221:218-225, 1996). In this study, further site-directed mutations were made at these positions within the M strain cDNA to determine whether the protein or nucleotide sequence directly controlled the symptom phenotype. The protein and not the nucleotide sequence directly controlled the symptom phenotype when amino acid 360 within the 126-kDa protein sequence was altered and likely controlled the symptom phenotype when amino acid 601 was altered. The effects of the substitutions at amino acid position 360 on viral protein accumulation were studied by pulse-labeling proteins in infected protoplasts. Accumulation of the 126- and 183-kDa proteins was less for an attenuated mutant than for two virulent mutants, but the viral movement protein and coat protein accumulated to levels reported to be sufficient for normal systemic symptom development. The size of necrotic local lesions on N. tabacum L. cv. Xanthi NN was negatively correlated with symptom development and accumulation of the 126-kDa protein for these mutants. With reference to this last finding, an explanation of the cause of the differing symptoms induced by these viruses is presented.

Tomato leaf curl geminivirus from India has a bipartite genome and coat protein is not essential for infectivity

Genomes of two isolates of tomato leaf curl geminivirus from India (ToLCV-India) have been sequenced. ToLCV-India contains A and B components, both of which are required for systemic movement and symptom development. The two isolates have 94% sequence identify but one isolate gave mild symptoms in Nicotiana benthamiana and tomato. The genome organization of ToLCV-India is similar to other whitefly-transmitted geminiviruses (WTGs) with bipartite genomes. However, it contains an additional ORF, AV3, that has not been reported for other WTGs. Its coat protein (CP) sequence is highly homologous to that of Indian cassava mosaic virus (90%). Two mutations that truncated the CP after amino acids 65 or 172 did not affect systemic movement and symptom development in either N. benthamiana or tomato. However, the symptoms caused by mutant viruses were different from those in plants infected with unmodified viruses, and plants infected with the mutants had markedly reduced amounts of single-stranded viral DNA. Comparison of sequences and other biological features of ToLCV-India with other geminiviruses showed that ToLCV-India is a distinct virus and is related to the WTGs from the Old World. It is similar to African cassava mosaic virus in its requirement for B component and dispensability of coat protein for symptom development, unlike other geminiviruses that infect tomato in the Old World. It is proposed that ToLCV-India evolved more recently as compared to other geminiviruses that infect tomato in the Old World.

The Kinetics of Infection of Zucchini Squash by Cucumber Mosaic Virus Indicate a Function for RNA 1 in Virus Movement

The differential rate of systemic symptom induction in zucchini squash by the Fny- and Sny-strains of cucumber mosaic virus (CMV) previously was mapped to RNA 1, which encodes a protein (1a) involved in virus replication. Examination of the kinetics of accumulation of the RNAs and the four encoded proteins in the inoculated cotyledons showed that the Fny-CMV-associated products generally appeared earlier then the Sny-CMV-associated products. In the systemically infected leaves, this difference was magnified, with a 2-day delay in the appearance of the Sny-CMV RNAs and encoded proteins. However, both Fny-CMV and Sny-CMV RNAs showed similar kinetics of RNA, 2a, 3a, and coat protein accumulation in protoplasts prepared from zucchini squash cotyledons. These data indicate that the differential rate of systemic symptom development was due to a difference in the rate of movement rather than the rate of replication. This was confirmed by a leaf-detachment assay, which showed a difference in the rate of systemic movement by Fny-CMV vs Sny-CMV, and by leaf-press blot hybridization of the inoculated cotyledons at different days postinoculation, which showed a difference in the rate of cell-to-cell movement by the two strains of CMV. Taken together, these data show that the rates of cell-to-cell and long-distance movement can be regulated by sequences in CMV RNA 1, previously thought to be involved only in virus replication.

Genetic engineering of potyvirus resistance using constructs derived from the zucchini yellow mosaic virus coat protein gene.

Three versions of the zucchini yellow mosaic virus (ZYMV) coat protein gene were engineered for expression in plants: the full-length coat protein sequence, the conserved core portion of the gene, and an antisense version. These constructs were introduced into muskmelon (Cucumis melo) and tobacco plants (Nicotiana tabacum) via Agrobacterium tumefaciens-mediated transformation; gene expression was verified by Northern and Western analysis. Transgenic R0 and R1 muskmelon plants expressing the full-length coat protein gene exhibited apparent immunity to ZYMV infection: There was a lack of symptom development during a 3-mo observation period and no measurable virus accumulation as determined by ELISA. Melon plants expressing the core or antisense constructs showed a several-day delay of systemic symptom development and reduction in virus titer. Furthermore, transgenic R1 tobacco plants expressing the full-length coat protein, core, or antisense constructs of ZYMV, a nonpathogen of tobacco, showed a short delay in symptom development and reduced virus titer when inoculated with the heterologous potyviruses, potato virus Y, and tobacco etch virus. The transgenic tobacco plants were not protected against the non-potyvirus, tobacco mosaic virus.