The <Emphasis Type="Italic">cyv-2</Emphasis> resistance to <Emphasis Type="Italic">Clover yellow vein virus</Emphasis> in pea is controlled by the eukaryotic initiation factor 4E

The same mutant allele of eukaryotic initiation factor 4E (eIF4E) that confers resistance to Pea seed-borne mosaic virus (sbm-1) and the white lupine strain of Bean yellow mosaic virus (wlv) also confers resistance to Clover yellow vein virus (ClYVV) in pea. The eIF4E genes from several pea lines were isolated and sequenced. Analysis of the eIF4E amino acid sequences from several resistant lines revealed that some lines, including PI 378159, have the same sequence as reported for sbm-1 and wlv. When eIF4E from a susceptible pea line was expressed from a ClYVV vector after mechanical inoculation of resistant PI 378159, the virus caused systemic infection, similar to its effects in susceptible line PI 250438. The resistance to ClYVV in line PI 378159 was characterized through a cross with PI 193835, which reportedly carries cyv-2. Mechanical inoculation of the F1 progeny with ClYVV resulted in no infection, indicating that the resistance gene in PI 378159 is identical to cyv-2 in PI 193835. Furthermore, particle bombardment of pea line PI 193835 with infectious cDNA of ClYVV (pClYVV/C3-S65T) resulted in the same resistance mode as that described for PI 378159. These results demonstrate that the resistance to ClYVV conferred by cyv-2 is mediated by eIF4E and that cyv-2 is identical to sbm-1 and wlv.     

Characterization of the recessive resistance gene cyv1 of Pisum sativum against Clover yellow vein virus

Two recessive resistance genes against Clover yellow vein virus (ClYVV), cyv1 and cyv2, have been previously reported. We recently screened resistant peas from a separate set of pea lines and classified them into two groups according to their distinct modes of resistance. We later revealed that one group carries cyv2, encoding eukaryotic translation initiation factor 4E (eIF4E), in linkage group (LG) VI. We explored the possibility that the resistance gene, tentatively designated non-cyv2, that confers resistance to the other group, was actually cyv1. We found that PI 236493, which carries cyv1, had restricted cell-to-cell movement of ClYVV similar to that in non-cyv2 peas including PI 429853. PI 429853 was crossed with susceptible line PI 250438. Mapping of F₂ progeny revealed that non-cyv2 was 4?cM from the simple sequence repeat marker AB40, whose loci are close to cyv1, mo, and sbm-2 mapped in LG II, which mediates resistance to other potyviruses. Moreover, PI 429853 crossed with PI 236493 produced F₁ progeny resistant to ClYVV, raising the possibility that non-cyv2 is allelic to cyv1. Because mo was previously mapped with eIF(iso)4E in LG II, we examined the possibility that non-cyv2, cyv1, and mo encoded eIF(iso)4E. However, there was no difference in the nucleotide sequence of the eIF(iso)4E-coding region between susceptible and resistant pea lines. The eIF(iso)4E gene was equivalently expressed in both PI 429853 and PI 250438 before and after ClYVV infection. Our results suggest that these resistance genes are unlikely to encode eIF(iso)4E on LG II.     

Genetic analysis of lethal tip necrosis induced by <Emphasis Type="Italic">Clover yellow vein virus</Emphasis> infection in pea

Clover yellow vein virus (ClYVV) elicits lethal tip necrosis in the pea line PI 118501. Pea line PI 118501 develops necrotic lesions and veinal necrosis on inoculated leaves, followed by systemic necrosis, leading to plant death. To understand the genetic basis of this lethal tip necrosis, we crossed lines PI 226564 and PI 250438, which develop mosaic symptoms in response to ClYVV inoculation. In reciprocal crosses of PI 118501 with PI 226564, all F₁ plants had mosaic symptoms with slight stem necrosis and early yellowing of upper leaves. Essentially the same symptom was manifested in PI 118501 × PI 250438 F₁ plants. In F₂ populations from the cross between PI 118501 and PI 226564, the observed ratios of necrosis, mosaic with slight stem necrosis, and mosaic fit the expected 1 : 2 : 1 ratio. These results indicate that a single incompletely dominant gene confers the induction of necrosis in PI 118501. This locus in pea, conferring necrosis induction to ClYVV infection, was designated Cyn1 (Clover yellow vein virus-induced necrosis). A linkage analysis using 100 recombinant inbred lines derived from a cross of PI 118501 and PI 226564 demonstrated that Cyn1 was located 7.5 cM from the SSR marker AD174 on linkage group III.     

Similarity of clover yellow vein virus and pea necrosis virus

There still is confusion concerning the relationships between clover yellow vein virus (ClYVV), pea necrosis virus (PNV) and bean yellow mosaic virus (BYMV). Therefore, three Swedish isolates of ClYVV and its type strain have now been compared with three isolates of PNV. A bean mosaic isolate and three pea necrosis isolates of BYMV have been used for reference. Based on host range tests, serology, and light microscope studies of inclusion bodies, ClYVV and PNV isolates are now considered to be strains of one virus, with the first name having priority. ClYVV (including the original PNV) especially differs from BYMV in its ability to infect white clover, to produce local lesions on cucumber cotyledons (at least two cultivars), to go systemic inChenopodium quinoa (the two local selections used at Wageningen and at Uppsala), to be rather virulent onNicotiana clevelandii, and to provoke extensive nucleolar enlargements in its host cells. Serologically the two viruses are more or less distinct.     

Advances and prospects in potato virology with special reference to virus resistance

Knowledge of the nucleotide sequences in the genomic nucleic acid of several potato viruses has enabled the open reading frames to be identified. These open reading frames are expressed by a variety of strategies, to produce proteins with functions in virus nucleic acid replication, virus particle production, cell-to-cell transport of virus and virus transmission by vectors. The activity of such proteins depends on their interactions with other viral or non-viral materials.Several other biological properties of plant viruses can also be related to individual viral gene products. For example, in plants co-infected with a specific pair of unrelated viruses, one virus can benefit from an ability to use the gene product of the second virus in replication, cell-to-cell transport or transmission by vectors. Similarly, different host resistance genes are targeted against viral replicase, movement protein or coat protein. Thus it is becoming possible to relate gene-for-gene (or more accurately, viral gene domain-host gene) interactions to events at the molecular level. Genetically engineered resistance to plant viruses likewise can be targeted against individual viral genes, and probably also against viral regulatory sequences. Such transgenic resistance seems likely to be as durable as conventional host resistance but durability should be improved by producing plants with combinations of resistances of different kinds, either conventional or genetically engineered, or both.     

The Use of Green Fluorescent Protein-Tagged Recombinant Viruses to Test Lettuce mosaic virus Resistance in Lettuce

ABSTRACT Seed certification and the use of cultivars containing one of two, probably allelic, recessive genes, mo1(1) and mo1(2), are the principal control methods for Lettuce mosaic virus (LMV) in lettuce. Although for a few LMV isolates, mo1(2) confers resistance with most isolates, the genes mo1(1) or mo1(2) confer a tolerance, and virus accumulation is readily detected in mo1-carrying plants. This phenotype complicates evaluation of the resistance status, in particular for mo1(1), for which there are no viral strains against which a true resistance is expressed. Two green fluorescent protein (GFP)-tagged viruses were constructed, derived from a non-resistance breaking isolate (LMV-0) and from a resistance-breaking isolate (LMV-E). An evaluation of 101 cultivars of known status was carried out with these recombinant viruses. Using the LMV-0-derived recombinant, identification of mo1-carrying cultivars was simple because, contrary to its wild-type parent, systemic movement of LMV-0-GFP was abolished in resistant plants. This assay detected four cases of misidentification of resistance status. In all these cases, further tests confirmed that the prior resistance status information was incorrect, so that a 100% correlation was observed between LMV-0-GFP behavior and the mo1 resistance status. Similarly, the LMV-E-derived recombinant allowed the identification of mo1(2) lettuce lines because its systemic movement was restricted in mo1(2) lines but not in susceptible or in mo1(1) lines. The tagged viruses were able to systemically invade another host, pea, irrespective of its resistance status against another member of the genus Potyvirus, Pea seed-borne mosaic virus. The use of these recombinant viruses could therefore greatly facilitate LMV resistance evaluation and speed up lettuce breeding programs.     

Resistance to Monopartite Begomoviruses Associated with the Bean Leaf Crumple Disease in Phaseolus vulgaris Controlled by a Single Dominant Gene

ABSTRACT Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Málaga virus are monopartite begomoviruses (genus Begomovirus, family Geminiviridae) that infect common bean (Phaseolus vulgaris), causing bean leaf crumple disease (BLCD). This disease was found to be widespread in southern Spain and causes stunted growth, flower abortion, and leaf and pod deformation in common bean plants. Commercial yield losses of up to 100% occur. In the present study, we have identified and characterized a resistance trait to BLCD-associated viruses in the common bean breeding line GG12. This resistance resulted in a complete absence of BLCD symptoms under field conditions or after experimental inoculation. Our analysis showed that virus replication was not inhibited. However, a severe restriction to systemic virus accumulation occurred in resistant plants, suggesting that cell-to-cell or long-distance movement were impaired. In addition, recovery from virus infection was observed in resistant plants. The reaction of P. vulgaris lines GG12 (resistant) and GG14 (susceptible), and of F(1), F(2), and backcross populations derived from them, to TYLCV inoculation suggested that a single dominant gene conferred the BLCD resistance described here.     

Signal Transduction in Resistance to Plant Viruses

Salicylic acid is part of a signal transduction pathway that induces resistance to viruses, bacteria and fungi. In tobacco and Arabidopsis the defensive signal transduction pathway branches downstream of salicylic acid. One branch induces PR-1 proteins and resistance to bacteria and fungi, while the other triggers induction of resistance to RNA and DNA viruses. This virus-specific branch can be activated using antimycin A and cyanide, or inhibited with salicylhydroxamic acid, suggesting a role for alternative oxidase in resistance to viruses. The virus-specific defensive pathway activates multiple resistance mechanisms. In tobacco, salicylic acid induces resistance to systemic movement of cucumber mosaic virus but has no effect on its replication or cell-to-cell movement. However, in the case of tobacco mosaic virus in tobacco, salicylic acid appears to induce interference with the synthesis of viral RNA.     

Transgenic resistance to cucumber mosaic virus in tomato: blocking of long-distance movement of the virus in lines harboring a defective viral replicase gene

ABSTRACT Tomato breeding lines were transformed with a defective replicase gene from RNA 2 of cucumber mosaic virus (CMV). A total of 63 transformants from five tomato genotypes were evaluated for resistance to CMV strains. The responses of R1 transgenic offspring fit into three categories: fully susceptible lines (44%), fully resistant lines (8%), and an intermediate-type mixture of susceptible and resistant seedlings in variable proportions (48%). Further characterization of the response of two highly resistant lines was performed by mechanical inoculation, aphid transmission, or grafting experiments. No virus was detected in noninoculated leaves from these lines, although a low level of virus accumulated initially in the inoculated leaf. The homozygous R2 plants and further generations that were evaluated (up to R5) showed resistance to the Fny-CMV strain, two Israeli isolates tentatively classified as subgroup IA, and K-CMV (a representative of subgroup IB). These lines were partially resistant to LS-CMV (a representative of subgroup II) when a high-virus-titer inoculum was used. Expression of the viral transgene was verified in these lines; however, the expected translation product was not detectable. In grafting experiments, we demonstrated that CMV virions were blocked in their ability to move from infected rootstocks of nontransformed tomato or tobacco into the transgenic scions. Interestingly, virions could not move through a transgenic intersection into the upper scion. These results provide an additional indication that replicase-mediated resistance affects long-distance movement.     

Cucumber mosaic virus Establishes Systemic Infection at Increased Temperature Following Viral Entrance Into the Phloem Pathway of Tetragonia expansa

ABSTRACT A potential regulatory site for Cucumber mosaic virus (CMV, pepo strain) movement necessary to establish systemic infection was identified through immunological and hybridization studies on Tetragonia expansa, which was systemically infected by CMV at 36 degrees C but not at 24 degrees C. In inoculated leaves, cell-to-cell movement of CMV was enhanced at 36 degrees C compared with that observed at 24 degrees C. CMV was distributed in the phloem cells of minor veins as well as epidermal and mesophyll cells at both 36 and 24 degrees C. CMV was detected in the petioles of inoculated leaves, stems, and petioles of uninoculated upper leaves at 36 degrees C, whereas CMV was detected only in the petioles of inoculated leaves and in stems at 24 degrees C. CMV moved into the phloem and was transported to the stem within 24 h postinoculation (hpi) at 36 degrees C. However, it did not accumulate in the petioles of the upper leaves until 36 hpi. In petioles of inoculated leaves at 24 degrees C, CMV was detected in the external phloem but not in the internal phloem. From these results, we conclude that systemic infection is established after viral entrance into the phloem pathway in T. expansa at 36 degrees C.     

Tissue culture methods for the screening and analysis of putative virus-resistant transgenic potato plants

ABSTRACT Following regeneration, putative virus-resistant transgenic plants are usually transferred from tissue culture to a greenhouse or growth chamber to screen for resistance to infection and disease development using mechanical, graft, or insect vector inoculation methods. To reduce initial screening costs and time, we developed mechanical and graft inoculation methods suitable for tissue culture use. The in vitro methods were validated by comparing them with similar greenhouse screens using putative potato virus Y strain o (PVY degrees ) replicase-mediated resistant regenerants of the potato cultivar Atlantic. Five transgenic lines were tested, with similar results obtained from in vitro and greenhouse experiments. Two of the transgenic lines, A1 and A3, showed the greatest resistance to PVY degrees infection, as indicated by low enzyme-linked immunosorbent assay values and infection rates. In vitro mechanical inoculation methods were also used to infect wild-type tomato and tobacco plants with cucumber mosaic virus and potato virus Y. Potato plants were also infected with the phloem-restricted potato leafroll virus, a low-titer virus, using in vitro graft inoculation methods. These results suggest the potential usefulness of these simple, effective, and economical techniques for screening large numbers of putative virus-resistant plants.     

Resistance to Plum pox virus in plants expressing cytosolic and nuclear single‐chain antibodies against the viral RNA NIb replicase

The expression of engineered single‐chain variable fragments specific to the NIb RNA replicase of Plum pox virus (PPV) (scFv2A) in transgenic plants was successfully used as a strategy to interfere with viral infection. Different scFv2A fusion proteins were constructed to target those subcellular compartments, such as the cytosol, endoplasmic reticulum (ER) membrane structures and the nucleus, where NIb protein presumably accumulates. Several transgenic lines of Nicotiana benthamiana plants expressing the scFv2A targeted to the cytosol (2A lines), ER (6K2 lines) and nucleus (NLS lines) were obtained. The protective effect of scFv expression was determined by mechanical virus inoculation in five 2A, three 6K2 and four NLS transgenic lines. The strongest resistance was afforded with the 2A‐3 (six non‐infected plants out of 10), 6K2‐1 (17 out of 33) and NLS‐11 (16 out of 19) transgenic lines. The success of this interference with PPV infection opens new possibilities for the control of this RNA virus and could be exploited not only to confer resistance in transgenic plants, but also to elucidate the role of the non‐structural NIb protein in different cell compartments during viral infection.     

Interference of Long-Distance Movement of Grapevine berry inner necrosis virus in Transgenic Plants Expressing a Defective Movement Protein of Apple chlorotic leaf spot virus

ABSTRACT Transgenic Nicotiana occidentalis plants expressing a movement protein (P50) and partially functional deletion mutants (DeltaA and DeltaC) of the Apple chlorotic leaf spot virus (ACLSV) showed resistance to Grapevine berry inner necrosis virus (GINV). The resistance is highly effective and GINV was below the level of detection in both inoculated and uninoculated upper leaves. In contrast, GINV accumulated in inoculated and uninoculated leaves of nontransgenic (NT) plants and transgenic plants expressing a dysfunctional mutant (DeltaG). On the other hand, in some plants of a transgenic plant line expressing a deletion mutant (DeltaA', deletion of the C-terminal 42 amino acids), GINV could spread in inoculated leaves, but not move into uninoculated leaves. In a tissue blot hybridization analysis of DeltaA'-plants inoculated with GINV, virus could be detected in leaf blade, midribs, and petiole of inoculated leaves, but neither in stems immediately above inoculated leaves nor in any tissues of uninoculated leaves. Immunohistochemical analysis of GINV-inoculated leaves of DeltaA'-plants showed that GINV could invade into phloem parenchyma cells through bundle sheath of minor veins, suggesting that the long-distance transport of GINV might be inhibited between the phloem cells and sieve element (and/or within sieve element) rather than bundle sheath-phloem interfaces. Immunogold electron microscopy using an anti-P50 antiserum showed that P50 accumulated on the parietal layer of sieve elements and on sieve plates. The results suggested that resistance in P50-transgenic plants to GINV is due to the interference of both long-distance and cell-to-cell movement of the virus.     

Defective Movement of Viruses in the Family Bromoviridae Is Differentially Complemented in Nicotiana benthamiana Expressing Tobamovirus or Dianthovirus Movement Proteins

ABSTRACT Taxonomically distinct tobacco mosaic tobamovirus (TMV), red clover necrotic mosaic dianthovirus (RCNMV), cucumber mosaic cucumovirus (CMV), brome mosaic bromovirus (BMV), and cowpea chlorotic mottle bromovirus (CCMV) exhibit differences in their host range. Each of these viruses encodes a functionally similar nonstructural movement protein (MP) that is essential for cell-to-cell movement of a progeny virus. Despite the lack of significant amino acid identity among the MPs of CMV, TMV, and RCNMV, movement-defective CMV (CMVFnyDeltaMP-DeltaKPN) was able to move locally and systemically in transgenic Nicotiana benthamiana expressing either TMV MP (NB-TMV-MP(+)) or RCNMV MP (NB-RCNMV-MP(+)). These observations contrast with those of previous studies in which transgenic N. tabacum cv. Xanthi plants expressing TMV MP supported only the cell-to-cell movement of CMVFnyDeltaMP-DeltaKPN. To verify whether similar complementation could be observed for movement-defective bromoviruses, NB-TMV-MP(+) and NB-RCNMV-MP(+) plants were inoculated independently with movement-defective variants of BMV (B3DeltaMP) and CCMV (CC3DeltaMP). Neither NB-TMV-MP(+) nor NB-RCNMV-MP(+) was able to rescue the defective cell-to-cell and long-distance movement of B3DeltaMP. In contrast, NB-RCNMV-MP(+) complemented the cell-to-cell, but not the long-distance, movement of CC3DeltaMP. Taken together, these studies suggest that virus movement is a complex process and that, in some cases, the host species plays a major role in determining the long-distance movement function of a virus.     

禾谷多粘菌分离、培养、接种及用于大麦抗黄花叶病的鉴定

 通过禾谷多粘菌Polymyxa graminis L.休眠孢子分离接种感病大麦品种,并进行砂培养,获得13个纯化了的禾谷多粘菌分离物,且其中3个带有大麦黄花叶病毒(BaYMV)。用分别带有BaYMV和大麦温和花叶病毒(BMMV)的英国禾谷多粘菌分离物的游动孢子接种13个中国大麦品种,以及用BaMMV摩擦接种36个中外大麦品种,抗性鉴定结果游动孢子接种与摩擦接种一样,均与田间鉴定结果一致,且大麦对BaYMV的抗性与对BaMMV的抗性一致,从而这2种接种方法可用于大麦品种(系)和育种中间体对BaYMV抗性的快速鉴定和筛选。游动孢子或休眠孢子接种方法还可有效地鉴定大麦对禾谷多粘菌的抗性。     

Unterschiedliche Formen der Resistenz gegen bodenbürtige Viren des Weizens

In Germany the furovirus Soil-borne cereal mosaic virus (SBCMV) and the bymovirus Wheat spindle streak mosaic virus (WSSMV) occur often together particularly in several rye production areas. Soil-borne wheat mosaic virus (SBWMV), a wheat infecting furovirus, has so far been found only in one field near Heidelberg. Each of these viruses is transmitted by Polymyxa graminis. The cultivation of resistant varieties is the only promising measure to prevent yield losses caused by soil-borne viruses. Resistance of wheat against the bymovirus WSSMV is comparable to the immunity of barley to the bymoviruses Barley yellow mosaic virus and Barley mild mosaic virus. In case of immunity no virus multiplication is observed in resistant cultivars. In contrast, all wheat cultivars are hosts of the furoviruses. All cultivars – including the resistant ones – can be infected following mechanical inoculation with SBWMV and SBCMV. Resistance to furoviruses is based on reduced levels of virus multiplication in roots and on inhibition of virus movement from roots to leaves. Because of the inhibited virus movement from roots to aerial parts of plants this type of resistance is referred to as translocation resistance. In spite of the different resistance mechanisms the absence of virus symptoms on the leaves is a common selection criterion for both immunity and translocation resistance. Therefore, the symptom free development of plants on uniformly infested fields is the best criterion for selecting wheat lines with resistance to soil-borne viruses. The limited suitability of other selection methods is discussed.     

豌豆病毒病病原研究

 1986年至1990年,从豌豆田中采集了150余份病毒病样本,鉴定出蚕豆萎蔫病毒(BB-WV)、芜菁花叶病毒(TuMV)、马铃薯Y病毒组分离物、黄瓜花叶病毒(CMV)、莴苣花叶病毒(LMV)、大豆花叶病毒(SMV)、豌豆花叶病毒(PMV)、菜豆黄花叶病毒(BYMV)和苜蓿花叶病毒(AMV)等9种病毒。样本中,BBWV所占的比例最高,达59.2%,其次为CMV,占15.5%。BBWV常与CMV复合侵染豌豆,LMV发生也较普遍。田间调查表明,豌豆病毒病发病率因种植地区及品种不同而有差异,平均发病率为12.4%。     

Identification and characterization of resistance to rhizomania in an ecotype of Beta vulgaris subsp. maritima

An ecotype (S023) of Beta vulgaris subsp. maritima was identified which was highly resistant to beet necrotic yellow vein virus (BNYVV) but susceptible to its fungal vector, Polymyxa betae . A comparative study of viral development in the roots showed that the resistance was effective from the early stages of infection. Mechanical inoculation experiments suggested that the resistance was not due to a lack of transmission of BNYVV by its fungal vector. Immunogold-silver labelling of the viral coat protein in root sections confirmed the existence of BNYVV transmission in the resistant ecotype. Virus replicated in isolated root protoplasts of both susceptible and resistant genotypes. Viral particles localized by immunogold-silver labelling diffused over short distances from the primary infected root cells in both resistant and susceptible plants. Evidence for inhibition of long-distance movement in the resistant ecotype requires further investigation.     

Evaluation of Maize Inbreds for Maize stripe virus and Maize mosaic virus Resistance: Disease Progress in Relation to Time and the Cumulative Number of Planthoppers

ABSTRACT Five tropical maize lines were tested and compared with the susceptible control line B73 for resistance to Maize stripe virus (MStV) and Maize mosaic virus (MMV), both propagatively transmitted by the planthopper Peregrinus maidis (Homoptera: Delphacidae). Resistance to each virus was evaluated separately by artificial inoculations with planthoppers viruliferous for either one virus or the other. Disease incidence and symptom severity progression were quantified in relation to time and the cumulative number of planthoppers. Line Hi40 was found to be susceptible to MStV and highly resistant to MMV. Generally, no MMV symptoms developed on Hi40, even under intense inoculation pressure by a large number of viruliferous planthoppers. Line Rev81 showed a partial but strong resistance to MStV, which mainly reduced disease incidence. Nevertheless, this resistance to MStV was the highest ever reported and held up, even when challenged by large numbers of planthoppers. The percentage of infected plants in line Rev81 never exceeded 30 to 40% in our experiments. Moderate levels of resistance to MStV, and to a lesser extent MMV, were found in lines 37-2, A211, and Mp705. However, resistance in these lines was completely overcome using a large number of insects transmitting either of the two viruses. These results suggest that different types of resistance to MMV and MStV are available in maize lines from Caribbean and Mascarene germ plasm. The expression of virus-specific resistance identified in Hi40 and Rev81 lines was not affected by intense inoculation pressure. In contrast, the moderate resistance in 37-2, A211, and Mp705 was partially effective against both viruses but not at high inoculation pressure. These different types of resistance, when present in the same genotype, could provide protection against both viruses.