Analysis of the autoproteolytic activity of the recombinant helper component proteinase from zucchini yellow mosaic virus

The multifunctional helper component proteinase (HC-Pro) of potyviruses contains an autoproteolytic function that, together with the protein 1 (P1) and NIa proteinase, processes the polyprotein into mature proteins. In this study, we analysed the autoproteolytic active domain of zucchini yellow mosaic virus (ZYMV) HC-Pro. Several Escherichia coli-expressed MBP:HC-Pro:GFP mutants containing deletions or point mutations at either the N- or C-terminus of the HC-Pro protein were examined. Our results showed that amino acids essential for the proteolytic activity of ZYMV HC-Pro are distinct from those of the tobacco etch virus HC-Pro, although the amino acid sequences in the proteolytic active domain are conserved among potyviruses.     

The conserved FRNK box in HC-Pro, a plant viral suppressor of gene silencing, is required for small RNA binding and mediates symptom development

The helper component-proteinase (HC-Pro) protein of potyviruses is a suppressor of gene silencing and has been shown to elicit plant developmental-defect-like symptoms. In Zucchini yellow mosaic virus (ZYMV), a mutation in the highly conserved FR₁₈₀NK box of HC-Pro to FI₁₈₀NK causes attenuation of these symptoms. At 5 days postinoculation and before symptoms appear, virus accumulation, HC-Pro protein levels, and viral short interfering RNA (siRNA) levels are similar for the severe (FRNK) and attenuated (FINK) strains. At this stage, ZYMV^FRNK caused greater accumulation of most microRNAs (miRNAs), and especially of their complementary miRNA “passenger” strands (miRNA*s), in systemically infected leaves than the attenuated ZYMV^FINK did. HC-Pro^FRNK specifically bound artificial siRNA and miRNA/miRNA* duplexes with a much higher affinity than the mutated HC-Pro^FINK. Further analysis of the mutant and wild-type HC-Pro proteins revealed that suppressor activity of the ZYMV HC^FINK mutant was not diminished. However, the FINK mutation caused a loss of HC-Pro suppressor function in other potyviruses. Replacement of the second positively charged amino acid in the ZYMV FRNK box to result in FRNA also caused symptom attenuation and reduced small RNA duplex-binding affinity without loss of suppressor activity. Our data suggest that the highly conserved FRNK box in the HC-Pro of potyviruses is a probable point of contact with siRNA and miRNA duplexes. The interaction of the FRNK box with populations of miRNAs directly influences their accumulation levels and regulatory functions, resulting in symptom development.     

Endophytic colonisation of squash by the fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales) for managing Zucchini yellow mosaic virus in cucurbits

We investigated the potential of endophytic Beauveria bassiana to provide protection against Zucchini yellow mosaic virus (ZYMV), one of the most economically important viral diseases in cucurbits. Four selected B. bassiana strains were able to successfully colonise squash plants following foliar inoculation with the conidial suspension of each respective strain. However, no significant difference in percentage colonisation was observed among the tested B. bassiana strains. Disease incidence (percentage of plants showing ZYMV symptoms) and severity (rating based on a 5-point scale), sampled weekly for four weeks following the challenge inoculation of plants with ZYMV, were significantly lower in B. bassiana-inoculated plants as compared to control plants, regardless of the inoculated strain. This is, to our knowledge, the first report on the potential of endophytic B. bassiana to confer protection against plant viruses. Further studies should be conducted to determine whether such endophytic B. bassiana-mediated protection against ZYMV in squash extends to other cucurbits.     

Apple stem-grooving virus: propagating hosts and purification

Isolates C₄₃₁ and DGEV of apple stem-grooving virus infected Zucchini squash (Cucurbita maxima) and pumpkin (C. pepo) systemically; these hosts were superior to Chenopodium quinoa for propagating the virus. Virus concentrations were assayed by counts of local lesions on cowpea (Vigna sinenis). Good yields of virus were concentrated from Zucchini squash sap clarified with bentonite or chloroform-bentonite and purified by densitygradient centrifugation and zone electrophoresis. Filamentous virus particles 615 nm (C₄₃₁) and 640 nm (DGEV), virtually free of host constituents and only slightly aggregated, were observed in electronmicrographs.     

Presence of Plant Viruses in some Rivers of Southern Italy

Water samples were collected from some rivers of Southern Italy whose water is normally used for irrigation and checked for the presence of plant viruses. The assayes were carried out by centrifuging at 5000x g one liter of water, resuspending the sediments in phosphate buffer and testing their infectivities on Chenopodium quinoa Willd. It was possible to isolate tobacco mosaic virus, cucumber mosaic virus and two more viruses not identified yet.     

Increase in Cucurbit Aphid–Borne Yellows Virus Concentration by Co–infection with Sap–transmissible Viruses does not Increase its Aphid Transmissibility

The cucurbit aphid–borne yellows virus (CABYV) is a new tentative member of the luteovirus group which is transmitted persistently by Myzus persicae and Aphis gossypii.
In muskmelon plants, mixed infection with CABYV and zucchini yellow mosaic virus (ZYMV) induced an increase in CABYV concentration estimated by double antibody sandwich–enzyme linked immunosorbent assay (DAS–ELISA), which was maximum after 3 weeks, of co–infection.
Assays, conducted with other cucurbit hosts and sap–transmissible viruses showed that a similar increase occurred with most of the potyviruses tested. However, cucumber mosaic virus (CMV) and squash mosaic virus (SqMV) were inefficient for less efficient than potyviruses) in increasing CABYV concentration.
Aphid transmission experiments were conducted to check whether increased virus multiplication could either enhance transmission rates or modify the mode of CABYV acquisition by aphids. However, when A. gossypii was used, no increases in CABYV, transmissibility nor in its acquisition mode were detected.     

Occurrence, distribution and relative incidence of mosaic viruses infecting field--grown squash in Tehran province, Iran

Squash (Cucurbita pepo) belongs to Cucurbitaceae family. Every year Cucurbitaceae are planted world wide. They are one of the most important economic crops. Cucurbitaceae are threatened by viruses. Many viruses damage the plants of this family. Since nine viruses have been reported on squash from Iran. In this survey, during 2002--2003, to determine the distribution of Cucumber mosaic virus (CMV), Zucchini yellow mosaic virus (ZYMV) and Watermelon mosaic virus (WMV), 466 samples were collected from squash field in Tehran province. Infected plants showing symptoms such as: mosaic, yellowing, deformation, shoestring of leaves and fruit deformation and yield reduction. Distribution of CMV, ZYMV and WMV were determined by DAS-ELISA. Thepercentage of ZYMV, WMV and CMV were 35.6, 26.1 and 25.1% respectively. Triple infection (CMV+ZYMV+WMV) were found in 6.4% of samples. ZYMV were found the most frequently the viruses. This is the first report of WMV on squash in Tehran province.     

Purification and properties of elderberry latent virus, one of four sap-transmissible viruses obtained from American elder (Sambucus canadensis L.)

Cherry leaf roll, tomato black ring and two previously unrecorded viruses were transmitted to Chenopodium quinoa from Sambucus canadensis plants imported from the U.S.A. Of the two newly recognized viruses one, code-named elderberry virus A, has filamentous particles about 650 times 15 nm; the other, named elderberry latent virus (ELV), was transmitted to several herbaceous species but remained symptomless in elder and most other hosts. In C. quinoa sap ELV lost infectivity after dilution to 10^--⁵ to 10^-- ⁶ , 10 min at 85–90°C, and 7 days at 18°C. Infectivity of nucleic acid extracts was abolished by ribonuclease in 0.2 m sodium chloride. ELV was purified from C. quinoa leaf extracts that were clarified with chloroform, by precipitation at pH 5 and differential centrifugation. Purified preparations contained numerous isometric particles c. 30 nm in diameter and a few particles c. 17 nm in diameter. In 0.06 M phosphate buffer ELV sedimented as a major 112 S (calculated for infinite dilution) component and a 48 S minor component. ELV showed no serological relationship to twenty-seven other isometric plant viruses. Its present cryptogram is R/I: *I*:S/S:S/*.     

Comparative Epidemiology of Three Virus Diseases on Zucchini Squash

Zucchini yellow mosaic virus (ZYMV), Papaya ringspot virus – type W (PRSV‐W) and Zucchini lethal chlorosis virus (ZLCV) cause important diseases on zucchini squash crops in Brazil. ZYMV and PRSV‐W belong to the genus Potyvirus and are transmitted by aphids, whereas ZLCV belongs to Tospovirus and is transmitted by the thrips Frankliniella zucchini. These three viruses may occur simultaneously in the field, and the epidemiology of the corresponding diseases may be determined by interactions among viruses, hosts and vectors. In this work, the progress of the diseases caused by these viruses was studied over a temporal and geographic range for three planting seasons (PS). For the lethal chlorosis (ZLCV), a monomolecular model was found to be the best fit for the data, though only during the third PS. For data collected during the first two PS, the Gompertz model was found to fit the data best. The spatial distribution of disease indicated disease aggregation at the end of the crop cycle. For the yellow mosaic (ZYMV), the model that best fit in the 1st PS was the logistic and in the 2nd and 3rd PS was monomolecular. The spatial pattern of the disease was random when the disease incidence was low but aggregated when the disease incidence was high. The common mosaic (PRSV‐W) showed the lowest incidence in all three PS. An exponential model was the best fit for data collected during all PS, and the spatial pattern of the disease was random. Interactions among the three viruses apparently did not result in changes in the epidemiology of the diseases. Removal of sources of inoculum and planting at an unfavourable time for reproduction of virus vectors are the two main measures recommended for the control of these diseases. The use of insecticide is indicated only for the control of the F. zucchini.     

Effects of green fluorescent protein or beta-glucuronidase tagging on the accumulation and pathogenicity of a resistance-breaking Lettuce mosaic virus isolate in susceptible and resistant lettuce cultivars

The RNA genome of a resistance-breaking isolate of Lettuce mosaic virus (LMV-E) was engineered to express the jellyfish green fluorescent protein (GFP) or beta-glucuronidase (GUS) fused to the helper-component proteinase (HC-Pro) to study LMV invasion and spread in susceptible and resistant lettuce cultivars. Virus accumulation and movement were monitored by either histochemical GUS assays or detection of GFP fluorescence under UV light. The GFP- and GUS-tagged viruses spread systemically in the susceptible lettuce cultivars Trocadero and Vanguard, where they induced attenuated symptoms, compared with the wild-type virus. Accumulation of the GFP-tagged virus was reduced but less affected than in the case of the GUS-tagged virus. Systemic movement of both recombinant viruses was very severely affected in Vanguard 75, a lettuce cultivar nearly isogenic to Vanguard but carrying the resistance gene mo1(2). Accumulation of the recombinant viruses in systemically infected leaves was either undetectable (GUS-tag) or erratic, strongly delayed, and inhibited by as much as 90% (GFP-tag). As a consequence, and contrary to the parental virus, the recombinant viruses were not able to overcome the protection afforded by the mo1(2) gene. Taken together, these results indicate that GUS or GFP tagging of the HC-Pro of LMV has significant negative effects on the biology of the virus, abolishing its resistance-breaking properties and reducing its pathogenicity in susceptible cultivars.     

Relative incidence,spatial distribution and genetic diversity of cucurbit viruses in eastern Spain

Viral diseases that could cause important economic losses often affect cucurbits, but only limited information on the incidence and spatial distribution of specific viruses is currently available. During the 2005 and 2006 growing seasons, systematic surveys were carried out in open field melon (Cucumis melo), squash and pumpkin (Cucurbita pepo), watermelon (Citrullus lanatus) and cucumber (Cucumis sativus) crops of the Spanish Community of Valencia (eastern Spain), where several counties have a long standing tradition of cucurbit cultivation and production. Surveyed fields were chosen with no previous information as to their sanitation status, and samples were taken from plants that showed virus‐like symptoms. Samples were analysed using molecular hybridisation to detect Beet pseudo‐yellows virus (BPYV), Cucurbit aphid‐borne yellows virus (CABYV), Cucumber mosaic virus (CMV), Cucumber vein yellowing virus (CVYV), Cucurbit yellow stunting disorder virus (CYSDV), Melon necrotic spot virus (MNSV), Papaya ring spot virus (PRSV), Watermelon mosaic virus (WMV) and Zucchini yellow mosaic virus (ZYMV). We collected 1767 samples from 122 independent field plots; out of these, approximately 94% of the samples were infected by at least one of these viruses. Percentages for the more frequently detected viruses were 35.8%, 27.0%, 16.5% and 7.2% for CABYV, WMV, PRSV and ZYMV, respectively, and significant deviations were found on the frequency distributions based on either the area or the host sampled. The number of multiple infections was high (average 36%), particularly for squash (more than 57%), with the most frequent combination being WMV + PRSV (12%) followed by WMV + CABYV (10%). Sequencing of WMV complementary DNA suggested that ‘emerging’ isolates have replaced the ‘classic’ ones, as described in southern regions of France, leading us to believe that cucurbit cultivation could be severely affected by these new, emerging isolates.     

Czech and scandinavian isolates resembling dandelion yellow mosaic virus

Virus isolates resembling the dandelion yellow mosaic virus (synonym: lettuce necrosis virus) were obtained from dandelion plants in twenty five localities of Bohemia and also of Norway, Sweden and Finland. All isolates were sap transmissible merely to lettuce, but some of them also toChenopodium quinoa; other test plants could not be infected. Attempted serological and biological identification of the isolates with some viruses presumed to be able to infect spontaneously dandelion plants have failed.     

Cell death triggered by the P25 protein in Potato virus X-associated synergisms results from endoplasmic reticulum stress in Nicotiana benthamiana

The synergistic interaction of Potato virus X (PVX) with a number of potyviruses results in systemic necrosis in Nicotiana spp. Previous investigations have indicated that the viral suppressor of RNA silencing (VSR) protein P25 of PVX triggers systemic necrosis in PVX-associated synergisms in a threshold-dependent manner. However, little is still known about the cellular processes that lead to this necrosis, and whether the VSR activity of P25 is involved in its elicitation. Here, we show that transient expression of P25 in the presence of VSRs from different viruses, including the helper component-proteinase (HC-Pro) of potyviruses, induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), which ultimately lead to ER collapse. However, the host RNA silencing pathway was dispensable for the elicitation of cell death by P25. Confocal microscopy studies in leaf patches co-expressing P25 and HC-Pro showed dramatic alterations in ER membrane structures, which correlated with the up-regulation of bZIP60 and several ER-resident chaperones, including the ER luminal binding protein (BiP). Overexpression of BiP alleviated the cell death induced by the potexviral P25 protein when expressed together with VSRs derived from different viruses. Conversely, silencing of the UPR master regulator, bZIP60, led to an increase in cell death elicited by the P25/HC-Pro combination as well as by PVX-associated synergism. In addition to its role as a negative regulator of P25-induced cell death, UPR partially restricted PVX infection. Thus, systemic necrosis caused by PVX-associated synergistic infections is probably the effect of an unmitigated ER stress following the overaccumulation of a viral protein, P25, with ER remodelling activity.     

Oxidising activity in root extracts from plants inoculated with virus or buffer that interferes with ELISA when using the substrate 3,3', 5,5'-tetramethylbenzidine

In indirect ELISA using protein A-horseradish peroxidase (HRP) as enzyme conjugate and 3,3′, 5,5′-tetramethylbenzidine (TMB) as substrate, extracts of roots of all cucumber, Chenopodium quinoa and Petunia hybrida plants previously inoculated with virus or buffer produced A₄₅₀ values up to seven-fold greater than those for comparable shoots or for extracts of roots from undisturbed, uninoculated plants, irrespective of the virus antiserum used for detection. This effect was also produced in tests in which no HRP conjugate was used, indicating that root extracts from virus-infected or physically injured plants, but not healthy uninjured plants, contain high levels of a factor able to oxidise TMB. The HRP conjugate/TMB substrate version of ELISA is therefore not reliable for detecting viruses in root extracts of herbaceous plants. In contrast, non-specific reactions were not obtained with root extracts, and viruses were reliably detected, when protein A-alkaline phosphatase was used as conjugate and p-nitrophenyl phosphate as substrate.     

Analysis of Tomato spotted wilt virus NSs protein indicates the importance of the N‐terminal domain for avirulence and RNA silencing suppression

Recently, Tomato spotted wilt virus (TSWV) nonstructural protein NSs has been identified unambiguously as an avirulence (Avr) determinant for Tomato spotted wilt (Tsw)‐based resistance. The observation that NSs from two natural resistance‐breaking isolates had lost RNA silencing suppressor (RSS) activity and Avr suggested a link between the two functions. To test this, a large set of NSs mutants was generated by alanine substitutions in NSs from resistance‐inducing wild‐type strains (NSs^RI), amino acid reversions in NSs from resistance‐breaking strains (NSs^RB), domain deletions and swapping. Testing these mutants for their ability to suppress green fluorescent protein (GFP) silencing and to trigger a Tsw‐mediated hypersensitive response (HR) revealed that the two functions can be separated. Changes in the N‐terminal domain were found to be detrimental for both activities and indicated the importance of this domain, additionally supported by domain swapping between NSs^RI and NSs^RB. Swapping domains between the closely related Tospovirus Groundnut ringspot virus (GRSV) NSs and TSWV NSs^RI showed that Avr functionality could not simply be transferred between species. Although deletion of the C‐terminal domain rendered NSs completely dysfunctional, only a few single‐amino‐acid mutations in the C‐terminus affected both functions. Mutation of a GW/WG motif (position 17/18) rendered NSs completely dysfunctional for RSS and Avr activity, and indicated a putative interaction between NSs and Argonaute 1 (AGO1), and its importance in TSWV virulence and viral counter defence against RNA interference.     

Comparative spatial spread overtime of Zucchini Yellow Mosaic Virus (ZYMV) and Watermelon Mosaic Virus (WMV) in fields of transgenic squash expressing the coat protein genes of ZYMV and WMV, and in fields of nontransgenic squash

The spatial and temporal patterns of aphid-vectored spread of Zucchini Yellow Mosaic Virus (ZYMV) and Watermelon Mosaic Virus (WMV) were monitored over two consecutive years in plantings of nontransgenic and transgenic squash ZW-20H (commercial cv. Freedom II) and ZW-20B, both expressing the coat protein genes of ZYMV and WMV. All test plants were surrounded by nontransgenic plants that were mechanically inoculated with ZYMV or WMV, and served as primary virus source. Across all trials, none of the transgenic plants exhibited systemic symptoms upon infection by ZYMV and WMV but a few of them developed localized chlorotic dots and/or blotches, and had low mixed infection rates [4% (6 of 139) of ZW-20H and 9% (13 of 139) of ZW-20B], as shown by ELISA. Geostatistical analysis of ELISA positive transgenic plants indicated, (i) a lack of spatial relationship on spread of ZYMV and WMV for ZW-20H with flat omnidirectional experimental semivariograms that fitted poorly theoretical models, and (ii) some extent of spatial dependence on ZYMV spread for ZW-20B with a well structured experimental semivariogram that fitted poorly theoretical models during the first but not the second growing season. In contrast, a strong spatial dependence on spread of ZYMV and WMV was found for nontransgenic plants, which developed severe systemic symptoms, had prevalent mixed infection rates (62%, 86 of 139), and well-defined omnidirectional experimental semivariograms that fitted a spherical model. Geostatistical data were sustained by virus transmission experiments with Myzus persicae in screenhouses, showing that commercial transgenic squash ZW-20H alter the dynamics of ZYMV and WMV epidemics by preventing secondary plant-to-plant spread.     

Studies on plant growth-regulating substances LV. The Plant growth-promoting properties of some β-aryl-propionic acids

Severe diseases of pepper (Capsicum annuum), tomato (Lycopersicon esculentum), eggplant (Solanum melongena) and tomato eggplant (Solanum integrifolium) in West Africa were induced by pepper veinal mottle virus (PVMV). Five selected virus isolates were serologically similar and readily transmissible by aphids in the non-persistent manner, but they differed in host range and/or symptoms induced in some susceptible species. One isolate from eggplant failed to infect pepper, Chenopodium quinoa and C. amaranticolor, and induced only local infections in tomato. An isolate from tomato failed to infect eggplant, and an isolate from tomato eggplant induced severe stunting in Physalis floridana. The type strain, like the isolate from tomato, failed to infect Nicotiana tabacum systemically, but each caused severe systemic leaf and stem necrosis in tomato. None of the isolates infected S. melongena cv. Long Purple, suggesting that PVMV might be controlled in this and perhaps other crop species by the use of immune or tolerant cultivars. All five isolates were serologically related to potato virus Y and some to six of 12 other potyviruses.     

Aphid-transmitted viruses from lilies in Britain

Three aphid-transmitted viruses, tulip breaking, lily symptomless and cucumber mosaic, were obtained from lilies in Britain. Tulip breaking virus was detected by the leaf mottle produced in Lilium formosanum, cucumber mosaic virus by inoculation of sap to Nicotiana clevelandii and Chenopodium quinoa, and lily symptomless virus by electron microscopy of crude leaf extracts from symptomless L. formosanum. Liiy symptomless virus was transmitted by Myzus persicae, Macrosiphum euphorbiae, Aulacorthum solani and Aphis fabae. M. persicae, which in a small experiment appeared a more efficient vector than A. fabae, transmitted the virus in a non-persistent manner. This conflicts with the original report of transmission in a persistent manner by A. gossypii but no transmission by M. persicae. The possibility that there are two distinct viruses with similar pathological effects is discussed. Tulip breaking and lily symptomless viruses spread to bait plants of L. formosanum within a field planting of lilies in Scotland especially during July to September; lily symptomless virus was the more prevalent. No spread of cucumber mosaic virus was detected.