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.     

Field testing for virus resistance and agronomic performance in transgenic plants

Resistance to specific virus diseases may be added as heritable characteristics to susceptible crop cultivars by transformation of the cultivars with specific virus-derived genes. In practice, however, resistance to the virus varies among transformed plants and transformation often changes cultivar yield and quality characteristics that are agronomically important. Therefore, rigorous selection among hundreds of different transformed plant lines is required to identify lines that are both virus resistant and also conform to or exceed standard characteristics of the original, susceptible cultivar. This paper describes methods we have developed for rapid selection of virus resistance, yield, and quality characteristics among transformed populations of plants.     

Breakdown of resistance to grapevine downy mildew upon limited deployment of a resistant variety

Background  

Natural disease resistance is a cost-effective and environmentally friendly way of controlling plant disease. Breeding programmes need to make sure that the resistance deployed is effective and durable. Grapevine downy mildew, caused by the Oomycete Plasmopara viticola, affects viticulture and it is controlled with pesticides. Downy mildew resistant grapevine varieties are a promising strategy to control the disease, but their use is currently restricted to very limited acreages. The arising of resistance-breaking isolates under such restricted deployment of resistant varieties would provide valuable information to design breeding strategies for the deployment of resistance genes over large acreages whilst reducing the risks of the resistance being defeated. The observation of heavy downy mildew symptoms on a plant of the resistant variety Bianca, whose resistance is conferred by a major gene, provided us with a putative example of emergence of a resistance-breaking isolate in the interaction between grapevine and P. viticola.     

Susceptibility of source plants to Sugarcane Fiji disease virus influences the acquisition and transmission of the virus by the planthopper vector Perkinsiella saccharicida

Abstract:  Fiji leaf gall (FLG) caused by Sugarcane Fiji disease virus (SCFDV) is transmitted by the planthopper Perkinsiella saccharicida . FLG is managed through the identification and exploitation of plant resistance. The glasshouse-based resistance screening produced inconsistent transmission results and the factors responsible for that are not known. A series of glasshouse trials conducted over a 2-year period was compared to identify the factors responsible for the erratic transmission results. SCFDV transmission was greater when the virus was acquired by the vector from a cultivar that was susceptible to the virus than when the virus was acquired from a resistant cultivar. Virus acquisition by the vector was also greater when the vector was exposed to the susceptible cultivars than when exposed to the resistant cultivar. Results suggest that the variation in transmission levels is due to variation in susceptibility of sugarcane cultivars to SCFDV used for virus acquisition by the vector.     

Symptom Severity, Yield, Seed Mottling and Seed Transmission of Soybean Mosaic Virus in Susceptible and Resistant Soybean: The Influence of Infection Stage and Growth Temperature

The effect of soybean mosaic virus (SMV) infection on symptom severity, yield, seed mottling and seed transmission in soybean in relation to the growth stage at infection and subsequent temperature was investigated using a susceptible (Harosoy), a moderately resistant (Evans) and a highly resistant (Merit) cultivar. Disease symptoms were more severe with early infection. A greater reduction in plant growth and seed yield, and higher percentages of mottled seeds and seed transmission of SMV also occurred with early infection. Virus titer was higher in younger plants than in older ones and also higher in plants infected at the ealier stage than at the later stage of growth. Merit (a highly resistant cultivar previously reported to be immune to seed mottling) inoculated at the early stage of plant growth resulted in infection and production of some mottled seeds. Temperature affected all parameters investigated. The effect of temperature was greater in the susceptible cultivar than in the resistant one. The optimal temperature for symptom severity, yield, seed mottling and seed transmission was 20 °C. Virus titer was highest at 30 °C in all three cultivars. Maturity of susceptible cultivar was delayed by infection.     

A novel hypersensitive resistance response against potato virus A in cultivar ’Shepody’

The potato cultivar ’Shepody’ is susceptible to a number of potato viruses including potato virus Y (PVY, potyvirus) but was found to possess extreme resistance to another potyvirus, potato virus A (PVA). ’Shepody’ plants were resistant to PVA infection in manual and graft inoculations. PVA replication was not detected in any of the inoculated plants by ELISA, an infectivity assay and RT-PCR. However, ’Shepody’ plants grafted with shoots containing PVA developed a novel symptomology which resembled a virus infection in appearance and in rate of translocation to the entire plant. Efforts to transmit the symptom-inducing agent manually failed. Graft-inoculation to potato virus indicator plants and PVA-susceptible potato plants showed that the symptom inducer was PVA at an extremely low concentration, detected using RT-PCR followed by Southern blot assay. Tubers from grafted but resistant ’Shepody’ plants had necrotic surfaces and internal spots. PVA was detected from necrotic areas but not from the non-necrotic ones. However, plants resulting from necrotic tubers were free from aerial leaf symptoms observed in grafted plants and produced non-necrotic normal tubers. A trace-back of the parental lineage of ’Shepody’ indicated that the resistance had been introgressed from the cultivar ’Bake King’. Analysis of progeny of a cross of resistant ’Shepody’ to the susceptible ’Goldrus’ indicated that this resistance is controlled by two independent dominant complementary genes in contrast to monogenic resistance reported for other potato viruses. Received: 6 April 1999 / Accepted: 28 July 1999     

Combining plant resistance and a natural enemy to control <Emphasis Type="Italic">Amphorophora idaei</Emphasis>

The European large raspberry aphid Amphorophora idaei Börner (Homoptera: Aphididae) is a virus vector of at least four plant virus complexes making it the most important aphid pest of raspberries in Northern Europe. An approach combining a bottom-up control (plant resistance) and a top-down control (an aphid parasitoid) using Aphidius ervi Haliday (Hymenoptera: Aphidiinae) was investigated in the laboratory. Aphid performance (pre-reproductive period, total reproductive output, lifespan and r _m) were compared when reared on both a susceptible cultivar and a resistant cultivar with significantly poorer performance on the resistant cultivar. Parasitoid attack behaviour increased with aphid density on both cultivars, but was significantly lower on resistant plants than susceptible plants. Aphids showed a greater tendency to drop from the plant when feeding on resistant plants compared with susceptible plants. The significance of the results is discussed in the context of possible control of the aphid using these combined methods.     

Genetic background matters: a plant-virus gene-for-gene interaction is strongly influenced by genetic contexts

Evolutionary processes responsible for parasite adaptation to their hosts determine our capacity to manage sustainably resistant plant crops. Most plant-parasite interactions studied so far correspond to gene-for-gene models in which the nature of the alleles present at a plant resistance locus and at a pathogen pathogenicity locus determine entirely the outcome of their confrontation. The interaction between the pepper pvr2 resistance locus and Potato virus Y (PVY) genome-linked protein VPg locus obeys this kind of model. Using synthetic chimeras between two parental PVY cDNA clones, we showed that the viral genetic background surrounding the VPg pathogenicity locus had a strong impact on the resistance breakdown capacity of the virus. Indeed, recombination of the cylindrical inclusion (CI) coding region between two PVY cDNA clones multiplied by six the virus capacity to break down the pvr2(3) -mediated resistance. High-throughput sequencing allowed the exploration of the diversity of PVY populations in response to the selection pressure of the pvr2(3) resistance. The CI chimera, which possessed an increased resistance breakdown capacity, did not show an increased mutation accumulation rate. Instead, selection of the most frequent resistance-breaking mutation seemed to be more efficient for the CI chimera than for the parental virus clone. These results echoed previous observations, which showed that the plant genetic background in which the pvr2(3) resistance gene was introduced modified strongly the efficiency of selection of resistance-breaking mutations by PVY. In a broader context, the PVY CI coding region is one of the first identified genetic factors to determine the evolvability of a plant virus.     

Tempo and mode of plant RNA virus escape from RNA interference-mediated resistance

A biotechnological application of artificial microRNAs (amiRs) is the generation of plants that are resistant to virus infection. This resistance has proven to be highly effective and sequence specific. However, before these transgenic plants can be deployed in the field, it is important to evaluate the likelihood of the emergence of resistance-breaking mutants. Two issues are of particular interest: (i) whether such mutants can arise in nontransgenic plants that may act as reservoirs and (ii) whether a suboptimal expression level of the transgene, resulting in subinhibitory concentrations of the amiR, would favor the emergence of escape mutants. To address the first issue, we experimentally evolved independent lineages of Turnip mosaic virus (TuMV) (family Potyviridae) in fully susceptible wild-type Arabidopsis thaliana plants and then simulated the spillover of the evolving virus to fully resistant A. thaliana transgenic plants. To address the second issue, the evolution phase took place with transgenic plants that expressed the amiR at subinhibitory concentrations. Our results show that TuMV populations replicating in susceptible hosts accumulated resistance-breaking alleles that resulted in the overcoming of the resistance of fully resistant plants. The rate at which resistance was broken was 7 times higher for TuMV populations that experienced subinhibitory concentrations of the antiviral amiR. A molecular characterization of escape alleles showed that they all contained at least one nucleotide substitution in the target sequence, generally a transition of the G-to-A and C-to-U types, with many instances of convergent molecular evolution. To better understand the viral population dynamics taking place within each host, as well as to evaluate relevant population genetic parameters, we performed in silico simulations of the experiments. Together, our results contribute to the rational management of amiR-based antiviral resistance in plants.     

Reversion of a resistance-breaking mutation shows reversion costs and high virus diversity at necrotic local lesions

An instance of host range evolution relevant to plant virus disease control is resistance breaking. Resistance breaking can be hindered by across-host fitness trade-offs generated by negative effects of resistance-breaking mutations on the virus fitness in susceptible hosts. Different mutations in pepper mild mottle virus (PMMoV) coat protein result in the breaking in pepper plants of the resistance determined by the L³ resistance allele. Of these, mutation M138N is widespread in PMMoV populations, despite associated fitness penalties in within-host multiplication and survival. The stability of mutation M138N was analysed by serial passaging in L³ resistant plants. Appearance on passaging of necrotic local lesions (NLL), indicating an effective L³ resistance, showed reversion to nonresistance-breaking phenotypes was common. Most revertant genotypes had the mutation N138K, which affects the properties of the virus particle, introducing a penalty of reversion. Hence, the costs of reversion may determine the evolution of resistance-breaking in addition to resistance-breaking costs. The genetic diversity of the virus population in NLL was much higher than in systemically infected tissues, and included mutations reported to break L³ resistance other than M138N. Infectivity assays on pepper genotypes with different L alleles showed high phenotypic diversity in respect to L alleles in NLL, including phenotypes not reported in nature. Thus, high diversity at NLL may potentiate the appearance of genotypes that enable the colonization of new host genotypes or species. Collectively, the results of this study contribute to better understanding the evolutionary dynamics of resistance breaking and host-range expansions.     

An eIF4E allele confers resistance to an uncapped and non-polyadenylated RNA virus in melon

The characterization of natural recessive resistance genes and virus-resistant mutants of Arabidopsis have implicated translation initiation factors of the 4E family [eIF4E and eIF(iso)4E] as susceptibility factors required for virus multiplication and resistance expression. To date, viruses controlled by these genes mainly belong to the family Potyviridae. Melon necrotic spot virus (MNSV) belongs to the family Tombusviridae (genus Carmovirus) and is an uncapped and non-polyadenylated RNA virus. In melon, nsv-mediated resistance is a natural source of recessive resistance against all strains of MNSV except MNSV-264. Analyses of chimeras between non-resistance-breaking and resistance-breaking strains have shown that the avirulence determinant maps to the 3'-untranslated region (3'-UTR) of the viral genome. Using a combination of positional cloning and microsynteny analysis between Arabidopsis thaliana and melon, we genetically and physically delimited the nsv locus to a single bacterial artificial chromosome clone and identified the melon eukaryotic translation initiation factor 4E (Cm-eIF4E) as a candidate gene. Complementation analysis using a biolistic transient expression assay, confirmed Cm-eIF4E as the product of nsv. A single amino acid change at position 228 of the protein led to the resistance to MNSV. Protein expression and cap-binding analysis showed that Cm-eIF4E encoded by a resistant plant was not affected in it's cap-binding activity. The Agrobacterium-mediated transient expression of the susceptibility allele of Cm-eIF4E in Nicotiana benthamiana enhanced MNSV-264 accumulation. Based on these results, a model to explain melon resistance to MNSV is proposed. These data, and data from other authors, suggest that translation initiation factors of the eIF4E family are universal determinants of plant susceptibility to RNA viruses.     

Location of the mechanism of resistance to Amphorophora agathonica (Hemiptera: Aphididae) in red raspberry

The aphid Amphorophora agathonica Hottes (Hemiptera: Aphididae) is an important virus vector in red (Rubus idaeus L.) and black (Rubus occidentalis L.) raspberries in North America. Raspberry resistance to A. agathonica in the form of a single dominant gene named Ag1 has been relied upon to help control aphid-transmitted plant viruses; however, the mechanism of resistance to the insect is poorly understood. Aphid feeding was monitored using an electrical penetration graph on the resistant red raspberry 'Tulameen' and compared with a susceptible control, 'Vintage'. There were no differences in pathway feeding behaviors of aphids as they moved toward the phloem. Once in the phloem, however, aphids feeding on resistant plants spent significantly more time salivating than on susceptible plants, and ingested significantly less phloem sap. This suggests that a mechanism for resistance to A. agathonica is located in the phloem. Reduced ingestion of phloem may result in inefficient acquisition of viruses and is a likely explanation for the lack of aphid-transmitted viruses in plantings of resistant cultivars.     

Enhanced sensitivity to higher ozone in a pathogen-resistant tobacco cultivar

Investigations of the effects of elevated ozone (O(3)) on the virus-plant system were conducted to inform virus pathogen management strategies better. One susceptible cultivar of tobacco (Nicotiana tabacum L. cv. Yongding) and a resistant cultivar (Nicotiana tabacum L. cv. Vam) to Potato virus Y petiole necrosis strain (PVY(N)) infection were grown in open-top chambers under ambient and elevated O(3) concentrations. Above-ground biomass, foliage chlorophyll, nitrogen and total non-structural carbohydrate (TNCs), soluble protein, total amino acid (TAA) and nicotine content, and peroxidase (POD) activity were measured to estimate the effects of elevated O(3) on the impact of PVY(N) in the two cultivars. Results showed that under ambient O(3), the resistant cultivar possessed greater biomass and a lower C/N ratio after infection than the susceptible cultivar; however, under elevated O(3), the resistant cultivar lost its biomass advantage but maintained a lower C/N ratio. Variation of foliar POD activity could be explained as a resistance cost which was significantly correlated with biomass and C/N ratio of the tobacco cultivar. Chlorophyll content remained steady in the resistant cultivar but decreased significantly in the susceptible cultivar when stressors were applied. Foliar soluble protein and free amino acid content, which were related to resistance cost changes, are also discussed. This study indicated that a virus-resistant tobacco cultivar showed increased sensitivity to elevated O(3) compared to a virus-sensitive cultivar.     

Influence of Soil Temperature on Meloidogyne incognita Resistant and Susceptible Cotton,Gossypium hirsutum

The degree of resistance by a cotton plant to Meloidogyne incognita is affected by soil temperature, particularly in moderately resistant cultivars, The total number of nematodes in the resistant and moderately resistant rools at 35 C was equal to, or greater than, the number in susceptible roots at 20, 25, or 30 C. A shift in numbers to developing and egg-bearing forms of nematodes in the susceptible cultivar as tentperature increased indicates development was affected by temperature rather than by genetic resistance mechanisms. However, the nematode resistant cultivar did not support maturation of nematodes until a soil tempurature of 35 C was attained. This indicated that resistance mechanisms are partially repressed at 35 C and differences in nematode development cannot be explained in terms of accumulated heat units. The moderately resistant cultivar was significantly more sensitive to the effects of high temperature than was the resistant cultivar.     

Interaction of Vesicular-Arbuscular Mycorrhizae and Cultivars of Alfalfa Susceptible and Resistant to Meloidogyne hapla

The interaction between vesicular-arbuscular mycorrhizal (VAM) fungi and the root-knot nematode (Meloidogyne hapla) was investigated using both nematode-susceptible (Grasslands Wairau) and nematode-resistant (Nevada Synthetic XX) cultivars of alfalfa (Medicago sativa) at four levels of applied phosphate. Mycorrhizal inoculation improved plant growth and reduced nematode numbers and adult development in roots in dually infected cultures of the susceptible cultivar. The tolerance of plants to nematode infection and development when preinfected with mycorrhizal fungi was no greater than when they were inoculated with nematodes and mycorrhizal fungi simultaneously. Growth of plants of the resistant cultivar was unaffected by nematode inoculation but was improved by mycorrhizal inoculation. Numbers of nematode juveniles were lower in the roots of the resistant than of the susceptible cultivar and were further reduced by mycorrhizal inoculation, although no adult nematodes developed in any resistant cultivar treatment. Inoculation of alfalfa with VAM fungi increased the tolerance and resistance of a cultivar susceptible to M. hapla and improved the resistance of a resistant cultivar.     

Combining transgenic and natural resistance to obtain broad resistance to tospovirus infection in tomato (Lycopersicon esculentum mill)

This study was undertaken to develop tomato plants with broad resistanceto tospoviruses which are a major limiting factor to tomato productionworldwide. A nontransgenic tomato line Stevens-Rodale (S-R), six transgenictomato lines expressing the nucleocapsid (N) protein gene of the lettuceisolate of tomato spotted wilt virus (TSWV-BL), and progeny of the crosses between S-Rand three of the transgenic lines homozygous for the N gene were evaluated fortheir resistance to tospovirus infection in greenhouse inoculation tests. S-Rhas the Sw-5 gene that confers resistance to several TSWVisolates. The six transgenic lines showed high levels of resistance wheninoculated with either TSWV-BL or a tomato isolate from Hawaii (TSWV-H).However, these same plants were highly susceptible to the Brazilian isolate ofgroundnut ringspot virus (GRSV-BR). Plants with the Sw-5gene were resistant to TSWV-BL and GRSV-BR, but were susceptible to TSWV-H.When inoculated with any of the three viruses, the F₁ progeny of thecrosses exhibited a susceptible, tolerant, or resistant phenotype with a higherproportion of the plants being either tolerant or resistant. When F₂progeny from F₁ resistant plants of each cross were inoculated withany of the three viruses, a higher proportion of tolerant and resistant plantswas observed compared to the F₁ progeny. Our results show thepotential to obtain broad resistance to tospoviruses by combining transgenicand natural resistance in a single plant.     

Comparative proteomic analysis of methyl jasmonate‐induced defense responses in different rice cultivars

Jasmonate is an important endogenous chemical signal that plays a role in modulation of plant defense responses. To understand its mechanisms in regulation of rice resistance against the fungal pathogen Magnaporthe oryzae, comparative phenotype and proteomic analyses were undertaken using two near‐isogenic cultivars with different levels of disease resistance. Methyl‐jasmonate (MeJA) treatment significantly enhanced the resistance against M. oryzae in both cultivars but the treated resistant cultivar maintained a higher level of resistance than the same treated susceptible cultivars. Proteomic analysis revealed 26 and 16 MeJA‐modulated proteins in resistant and susceptible cultivars, respectively, and both cultivars shared a common set of 13 proteins. Cumulatively, a total of 29 unique MeJA‐influenced proteins were identified with many of them known to be associated with plant defense response and ROS accumulation. Consistent with the findings of proteomic analysis, MeJA treatment increased ROS accumulation in both cultivars with the resistant cultivar showing higher levels of ROS production and cell membrane damage than the susceptible cultivar. Taken together, our data add a new insight into the mechanisms of overall MeJA‐induced rice defense response and provide a molecular basis of using MeJA to enhance fungal disease resistance in resistant and susceptible rice cultivars.     

Responses of some Asian and European barley cultivars to UK and Chinese isolates of soil-borne barley mosaic viruses

In field plots at Yancheng, Jiangsu, China, a range of European and Asian barley cultivars was grown in soil from three sites in China infested with barley yellow mosaic virus (BaYMV). Most of the cultivars resistant to the common European strain of BaYMV were susceptible to the Chinese isolates but cv. Energy remained disease-free. Barley mild mosaic virus (BaMMV) was also detected in one of these soils but affected only one Chinese cultivar and not those susceptible to BaMMV in Europe. This is the first report of BaMMV in China. Inoculation experiments confirmed the different cultivar response to UK and Chinese isolates of BaYMV and showed that resistance was to the virus and not to the vector. A range of Chinese cultivars selected for resistance to BaYMV were also resistant to a UK isolate of BaMMV.     

A natural recessive resistance gene against potato virus Y in pepper corresponds to the eukaryotic initiation factor 4E (eIF4E)

We show here that the pvr2 locus in pepper, conferring recessive resistance against strains of potato virus Y (PVY), corresponds to a eukaryotic initiation factor 4E (eIF4E) gene. RFLP analysis on the PVY-susceptible and resistant pepper cultivars, using an eIF4E cDNA from tobacco as probe, revealed perfect map co-segregation between a polymorphism in the eIF4E gene and the pvr2 alleles, pvr2(1) (resistant to PVY-0) and pvr2(2) (resistant to PVY-0 and 1). The cloned pepper eIF4E cDNA encoded a 228 amino acid polypeptide with 70-86% nucleotide sequence identity with other plant eIF4Es. The sequences of eIF4E protein from two PVY-susceptible cultivars were identical and differed from the eIF4E sequences of the two PVY-resistant cultivars Yolo Y (YY) (pvr2(1)) and FloridaVR2 (F) (pvr2(2)) at two amino acids, a mutation common to both resistant genotypes and a second mutation specific to each. Complementation experiments were used to show that the eIF4E gene corresponds to pvr2. Thus, potato virus X-mediated transient expression of eIF4E from susceptible cultivar Yolo Wonder (YW) in the resistant genotype YY resulted in loss of resistance to subsequent PVY-0 inoculation and transient expression of eIF4E from YY (resistant to PVY-0; susceptible to PVY-1) rendered genotype F susceptible to PVY-1. Several lines of evidence indicate that interaction between the potyvirus genome-linked protein (VPg) and eIF4E are important for virus infectivity, suggesting that the recessive resistance could be due to incompatibility between the VPg and eIF4E in the resistant genotype.