A single amino Acid change in viral genome-associated protein of potato virus y correlates with resistance breaking in 'virgin a mutant' tobacco

ABSTRACT Tobacco cultivar Virgin A Mutant (VAM) is reported to have the recessive potyvirus resistance gene va. Varied levels of resistance were observed in VAM plants inoculated with Japanese potato virus Y (PVY) isolates. VAM was highly resistant to most of the PVY isolates tested and tolerant to three necrotic strain isolates of PVY-T. Based on data obtained from tissue printing and press blotting, the resistance appeared to be mainly at the level of cell-to-cell movement. PVY replicated in VAM proto-plasts, but the replication was 30% lower than in susceptible tobacco, suggesting that impairment of replication also contributes to resistance. To identify the viral gene product or products involved in VAM resistance, we isolated spontaneous resistance-breaking mutants by passing vein-banding (O strain) isolates several times through VAM plants. By comparing the amino acid sequences of the mutants with their original isolates, we identified a single amino acid substitution in the viral genome-associated protein (VPg) domain that is correlated with VAM resistance breaking. Together, these results suggest that, in addition to its role in replication, VPg plays an important role in the cell-to-cell movement of PVY.     

靶向G-四链体的抗番茄丛矮病毒卟啉类衍生物的筛选

植物病毒病给蔬菜生产带来很大影响,目前蔬菜病毒防控主要采用非药剂措施,广谱抗病毒剂效果有限,需要基于新的药物靶点开发新的病毒抑制剂。G-四链体(G4s)是一种特殊的核酸高级结构,其在病毒基因组中的形成或解链可调节基因的复制、转录和翻译等过程,进而影响病毒增殖。一些可调节G-四链体结构稳定性的小分子呈现出抗病毒活性,使得G-四链体有望成为新的抗病毒药物的靶标。番茄丛矮病毒(tomato bushy stunt virus, TBSV)是一种分布广泛的植物病毒,有关其基因组的复制、转录和翻译等分子机制已研究得较为成熟,是一种研究病毒与寄主互作的模式病毒。揭示TBSV中G-四链体的结构及功能,有望为植物病毒基因中G-四链体的研究奠定基础。本研究通过生物信息学分析,在TBSV基因中鉴定出两条保守的、潜在的G-四链体可形成序列(putative G-quadruplex sequences, PQS)——TBSV-PQS2和TBSV-PQS4;通过紫外、荧光光谱和圆二色光谱(CD光谱)方法筛选出与TBSV-PQS2和TBSV-PQS4互作的G4配体;通过烟草体内侵染性克隆试验发现,G-四链体配体...     

Two Resistance Modes to Clover yellow vein virus in Pea Characterized by a Green Fluorescent Protein-Tagged Virus

ABSTRACT This study characterized resistance in pea lines PI 347295 and PI 378159 to Clover yellow vein virus (ClYVV). Genetic cross experiments showed that a single recessive gene controls resistance in both lines. Conventional mechanical inoculation did not result in infection; however, particle bombardment with infectious plasmid or mechanical inoculation with concentrated viral inocula did cause infection. When ClYVV No. 30 isolate was tagged with a green fluorescent protein (GFP) and used to monitor infection, viral cell-to-cell movement differed in the two pea lines. In PI 347595, ClYVV replicated at a single-cell level, but did not move to neighboring cells, indicating that resistance operated at a cell-to-cell step. In PI 378159, the virus moved to cells around the infection site and reached the leaf veins, but viral movement was slower than that in the susceptible line. The viruses observed around the infection sites and in the veins were then recovered and inoculated again by a conventional mechanical inoculation method onto PI 378159 demonstrating that ClYVV probably had mutated and newly emerged mutant viruses can move to neighboring cells and systemically infect the plants. Tagging the virus with GFP was an efficient tool for characterizing resistance modes. Implications of the two resistance modes are discussed.     

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.     

A transgenic RNAi approach for developing tomato plants immune to Pepino mosaic virus

RNA interference (RNAi) or gene silencing is a natural defence response of plants to invading viruses. Here, we applied this approach against pepino mosaic virus (PepMV) isolates in their natural host, tomato. PepMV isolates differ in their genetic sequences, the severity of the disease they induce, and their worldwide distribution. PepMV causes heavy crop losses, mainly due to impaired tomato fruit quality. Resistant varieties are not yet available, despite many years of resistance breeding efforts within the tomato seed industry. To generate broad resistance to PepMV strains, conserved sequences from three different strains of PepMV (US1, LP, and CH2) were synthesized as a single insert and cloned in a hairpin configuration into a binary vector, which was used to transform tomato plants. Transgenic tomato lines that expressed a high level of transgene-siRNA exhibited immunity to PepMV strains, including a new Israeli isolate. This immunity was maintained even after graft inoculation, in which a transgenic scion was grafted onto nontransgenic infected rootstocks. However, an immune transgenic rootstock was unable to induce resistance in a nontransformed scion. These results provide the first example of engineered immunity to diverse PepMV strains in transgenic tomato based on gene silencing.     

Spinach curly top virus: A Newly Described Curtovirus Species from Southwest Texas with Incongruent Gene Phylogenies

ABSTRACT A curtovirus associated with a disease of spinach was isolated in southwest Texas during 1996. Disease symptoms included severe stunting and chlorosis, with younger leaves curled, distorted, and dwarfed. Viral DNA was purified and an infectious clone obtained. Agroinoculation using a construct bearing full-length tandem repeats of the cloned viral genome resulted in systemic infection of species in six of seven plant families tested, indicating that the virus has a wide host range. Symptoms produced in spinach agroinoculated with cloned viral DNA were similar to those observed in the field. Viral single-stranded and double-stranded DNA forms typical of curtovirus infection were detected in host plants by Southern blot hybridization. The complete sequence of the infectious clone comprised 2,925 nucleotides, with seven open reading frames encoding proteins homologous to those of other curtoviruses. Complete genome comparisons revealed that the spinach curtovirus shared 64.2 to 83.9% nucleotide sequence identity relative to four previously characterized curtovirus species: Beet curly top virus, Beet severe curly top virus, Beet mild curly top virus, and Horseradish curly top virus. Phylogenetic analysis of individual open reading frames indicated that the evolutionary history of the three virion-sense genes was different from that of the four complementary-sense genes, suggesting that recombination among curtoviruses may have occurred. Collectively, these results indicate that the spinach curtovirus characterized here represents a newly described species of the genus Curtovirus, for which we propose the name Spinach curly top virus.     

Extreme Reduction of Disease in Oats Transformed with the 5' Half of the Barley Yellow Dwarf Virus-PAV Genome

ABSTRACT Barley yellow dwarf viruses (BYDVs) are the most serious and widespread viruses of oats, barley, and wheat worldwide. Natural resistance is inadequate. Toward overcoming this limitation, we engineered virus-derived transgenic resistance in oat. Oat plants were transformed with the 5' half of the BYDV strain PAV genome, which includes the RNA-dependent RNA polymerase gene. In experiments on T2- and T3-generation plants descended from the same transformation event, all BYDV-inoculated plants containing the transgene showed disease symptoms initially, but recovered, flowered, and produced seed. In contrast, all but one of the BYDV-PAV-inoculated nontransgenic segregants died before reaching 25 cm in height. Although all of the recovered transgenic plants looked similar, the amount of virus and viral RNA ranged from substantial to undetectable levels. Thus, the transgene may act either by restricting virus accumulation or by a novel transgenic tolerance phenomenon. This work demonstrates a strategy for genetically stable transgenic resistance to BYDVs that should apply to all hosts of the virus.     

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.     

Near immunity to rice tungro spherical virus achieved in rice by a replicase-mediated resistance strategy

ABSTRACT Rice tungro disease is caused by rice tungro bacilliform virus (RTBV), which is responsible for the symptoms, and rice tungro spherical virus (RTSV), which assists transmission of both viruses by leafhoppers. Transgenic japonica rice plants (Oryza sativa) were produced containing the RTSV replicase (Rep) gene in the sense or antisense orientation. Over 70% of the plants contained one to five copies of the Rep gene, with integration occurring at a single locus in most cases. Plants producing antisense sequences exhibited significant but moderate resistance to RTSV (60%); accumulation of antisense RNA was substantial, indicating that the protection was not of the homology-dependent type. Plants expressing the full-length Rep gene, as well as a truncated Rep gene, in the (+)-sense orientation were 100% resistant to RTSV even when challenged with a high level of inoculum. Accumulation of viral RNA was low, leading us to conclude that RTSV Rep-mediated resistance is not protein-mediated but is of the cosuppression type. Resistance was effective against geographically distinct RTSV isolates. In addition, RTSV-resistant transgenic rice plants were unable to assist transmission of RTBV. Such transgenic plants could be used in an epidemiological approach to combat the spread of the tungro disease.     

Turnip crinkle virus with nonviral gene cancels the effect of silencing suppressors of P19 and 2b in Arabidopsis thaliana

In plants, green fluorescent protein (GFP) has become a preferred molecular marker for gene expression and cellular localization, and plant viral vectors are valuable tools for heterologous gene expression. Some plant viruses have been used for expression of GFP, and the activities of these viruses are barely affected by the extra GFP gene. In contrast, the packaging and the length of Turnip crinkle virus (TCV) genome is strictly limited when foreign genes are inserted into the coding sequences of TCV genome. In this report, we removed the silencing suppressor p38 from TCV, and constructed GFP derivatives of TCV. Then the resulting TCV mutants were used to infect Arabidopsis plants containing mutations in key silencing pathway genes, including triple dcl2/dcl3/dcl4, dcl2, dcl4 and ago mutant plants. Our results demonstrate that the activity of TCV is affected by nonviral GFP insert in Arabidopsis plants, and RNA silencing appears not play an important role. AGOs appear to be more efficient at slicing RNAs of viral origin, especially AGO2 and AGO7. Although the viral suppressors of RNA silencing (VSRs) P19 and 2b can enhance the accumulation of viral RNAs, neither P19 nor 2b can significantly increase the expression of TCV mutants with nonviral genes. TCV is an example of an RNA virus that is recalcitrant to add nonviral gene sequences.     

Virus recognition and pathogenicity: Implications for resistance mechanisms and breeding

The major method of control of virus diseases in crop plants is breeding for resistance. The genetics of resistance, and of matching virulence (the ability of a virus strain to overcome a specific host resistance gene) have been studied less for viruses than for fungal and bacterial pathogens. This paper draws on a survey of the genetics of resistance to a large number of viruses in cultivated crops, and makes some generalisations and predictions about mechanisms. Most resistance to viruses in crops is monogenic. Dominant alleles are associated with virus-localisation mechanisms, which are induced after infection. The nature of the ‘recognition event’ between plant- and virus-coded functions, which triggers resistance plus a cascade of secondary responses, is not yet known. Gene dosage-dependent alleles tend to be associated with non-localising resistance, which allows some virus spread, but inhibits multiplication. Recessive alleles may involve a negative type of resistance mechanism, whereby the resistant plant lacks some function normally required by the virus for pathogenesis. Such resistance tends to be expressed as complete immunity. Many resistance genes have been overcome by virulent isolates of viruses; only 10 % of the sample of resistance genes have proved exceptionally durable. Virulence may involve different viral functions. The production of infectious cDNA clones, and construction of chimaeric recombinants between clones of virulent and avirulent isolates, is now allowing detailed mapping of virulence determinants. Transformation of plants with ‘novel’ genes for virus resistance, based on coat proteins and viral satellites, may allow construction of more robust resistance systems.     

The use of Plum pox virus as a plant expression vector

Among the biotechnological uses of plant viruses, the expression of foreign sequences through virus-based vectors represents a promising research area. The potyvirus Plum pox virus (PPV) has been used to design expression vectors which have allowed successful expression of foreign sequences in plants, either in the form of small peptides fused to the viral coat protein, or as whole independent proteins inserted on different points of the genome. The present review describes the different PPV-based vectors that have been produced, including information regarding relevant aspects of their use, such as the optimal location of peptides or the stability of inserts. Recent developments, like the expression of proteins on stone fruit trees by using PPV-based vectors capable to infect woody plants, are also described.     

Positional effect of gene insertion on genetic stability of a clover yellow vein virus-based expression vector

The stability of the inserted genes in the viral expression vector varied depending on the sequence introduced and the position of insertion. Infectious cDNA to Clover yellow vein virus (pClYVV) was modified to insert a foreign gene at two independent sites: one, along with a polylinker, between the NIb and CP genes (pClYVV/CP/W) and the other between P1 and HC-Pro (pClYVV-Pst/CP). The green fluorescent protein (GFP) gene was inserted into either pClYVV/CP/W or pClYVV-Pst/CP. GFP gene was stably maintained and expressed in both vectors following serial passages in plants. Progeny viruses from both constructs accumulated in similar amounts and at rates of 70%–80% of that of the wild-type virus. On the other hand, progeny viruses carrying the human interferon- (hIFN) gene cloned in pClYVV-Pst/CP were genetically unstable owing to frequent deletions of the cloned gene during passage through plants. In contrast, the hIFN sequence cloned in pClYVV/CP/W was stably maintained in viruses after several passages in broad bean plants, and the progeny virus accumulated at the rate of about 50%–100% of that of the wild-type virus. The nucleotide sequence analyses indicated that the genetic instability of the inserted sequence results from homologous recombination of viral vector and inserted DNA sequences; it is not due to the inserted sequence alone.     

Host factors used by positive-strand RNA plant viruses for genome replication

Replication of positive-strand RNA [(+)RNA] viruses proceeds through well-orchestrated actions of both viral and host factors. Remarkable features of eukaryotic (+)RNA virus replication include hijacking of host factors by viral components and remodeling of intracellular membranes to establish the viral replication factory, where viral RNA is synthesized. Here we review recent progress in our understanding of how (+)RNA plant viruses use host factors to create favorable environments for viral RNA replication.     

蚜虫传播非持久性病毒的取食行为调控机制

蚜虫能够传播上百种植物病毒，是最重要的农业介体昆虫之一。蚜虫在刺探和取食植物过程中，唾液组分会连同附着在口针中的病毒粒子一同被分泌进入植物内，在调节植物诱导抗性、病毒侵染扩散、介体昆虫行为等过程中均有重要作用。本文围绕蚜虫传播病毒和获取病毒2个关键过程，总结分析了蚜虫独特的刺吸取食行为与传毒效率和获毒效率之间的联系；针对取食活动中关键的唾液蛋白在调控植物免疫抗性、帮助病毒侵染过程中的功能，阐述了蚜虫高效传播非持久病毒的分子基础；针对蚜虫的获毒过程，综述了病毒侵染植物间接调控蚜虫趋向和行为的作用方式。这些研究的开展将为解释蚜虫和病毒协同侵染的分子机制以及有效开展基于蚜虫取食行为调控的病虫害防控新技术提供思路。     

Biological and molecular characterization of a novel carmovirus isolated from angelonia

ABSTRACT A new carmovirus was isolated from Angelonia plants (Angelonia angustifolia), with flower break and mild foliar symptoms, grown in the United States and Israel. The virus, for which the name Angelonia flower break virus (AnFBV) is proposed, has isometric particles, approximately 30 nm in diameter. The experimental host range was limited to Nicotiana species, Schizanthus pinnatus, Myosotis sylvatica, Phlox drummondii, and Digitalis purpurea. Virions were isolated from systemically infected N. benthamiana leaves, and directly from naturally infected Angelonia leaves, using typical carmovirus protocols. Koch's postulates were completed by mechanical inoculation of uninfected Angelonia seedlings with purified virions. Isometric particles were observed in leaf dips and virion preparations from both Angelonia and N. benthamiana, and in thin sections of Angelonia flower tissue by electron microscopy. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis of dissociated purified virus preparations, a major protein component with a molecular mass of 38 kDa was observed. Virion preparations were used to produce virus-specific polyclonal antisera in both Israel and the United States. The antisera did not react with Pelargonium flower break virus (PFBV), Carnation mottle virus (CarMV), or Saguaro cactus virus (SgCV) by either enzyme-linked immunosorbent assay or immunoblotting. In reciprocal tests, antisera against PFBV, CarMV, and SgCV reacted only with the homologous viruses. The complete nucleotide sequence of a Florida isolate of AnFBV and the coat protein (CP) gene sequences of Israeli and Maryland isolates were determined. The genomic RNA is 3,964 nucleotides and contains four open reading frames arranged in a manner typical of carmoviruses. The AnFBV CP is most closely related to PFBV, whereas the AnFBV replicase is most closely related to PFBV, CarMV, and SgCV. Particle morphology, serological properties, genome organization, and phylogenetic analysis are all consistent with assignment of AnFBV to the genus Carmovirus.     

Molecular breeding for virus resistant potato plants

To engineer resistance against potato virus X (PVX), the viral coat protein (CP) gene has been introduced into two potato cultivars. Stable expression of the gene in transgenic clones throughout the growing season has been obtained and resulted in considerably increased virus resistance. With varying frequencies depending on the original cultivar used, true-to-type PVX resistant transgenic clones have been obtained. Since deviant light sprout characteristics were invariably associated with aberrations in plant phenotype, they can be used in procedures to early screen for deviations. Furthermore, it has been possible to unequivocally discriminate between the original untransformed and independent transgenic cultivars. Although no relation has been found between the presence, if any, of the CP of potato virus Y (PVY) or potato leafroll virus (PLRV) in CP gene transgenic potato, appreciable levels of resistance to these viruses has been obtained. This suggests that the mechanism by which a viral CP gene in the potato genome evokes resistance, differs amongst various viruses.