Amino acid changes in P3, and not the overlapping pipo-encoded protein, determine virulence of soybean mosaic virus on functionally immune Rsv1-genotype soybean

A small open reading frame, termed 'pipo', is embedded in the P3 cistron of potyviruses. Currently, knowledge on pipo and its role(s) in the life cycle of potyviruses is limited. The P3 and helper-component proteinase (HC-Pro) cistrons of Soybean mosaic virus (SMV) harbour determinants affecting virulence on functionally immune Rsv1-genotype soybeans. Interestingly, a key virulence determinant of SMV on Rsv1-genotype soybeans (i.e. soybeans containing the Rsv1 resistance gene) that resides at polyprotein codon 947 overlaps both P3 and a pipo-encoded codon. This raises the question of whether PIPO or P3 is the virulence factor. To answer this question, the corresponding pipo of an avirulent and two virulent strains of SMV were studied by comparative genomics, followed by syntheses and analyses of site-directed mutants. Our data demonstrate that the virulence of SMV on Rsv1-genotype soybeans is affected by P3 and not the overlapping pipo-encoded protein.     

大豆花叶病毒半夏分离物侵染性克隆构建及鉴定

大豆花叶病毒(Soybean mosaic virus,SMV)作为半夏(Pinellia ternata (Thunb.) Breit.)主要病毒病害之一,已对其产量和品质造成严重影响。构建病毒侵染性克隆是反向遗传学研究病毒基因功能、病毒与宿主相互作用的有力工具,为明确SMV侵染半夏的分子机制,开展SMV全长cDNA侵染性克隆的构建特别重要。因此文中利用Gibson体外重组系统对大豆花叶病毒山西半夏分离物(SMV-SXBX)侵染性克隆进行组装,通过农杆菌浸润法接种健康半夏;进一步通过机械传代、逆转录-聚合酶链式反应(RT-PCR)证实SMV-SXBX侵染性克隆3′末端含有poly(A)尾56 nt时具有稳定侵染性。该方法便捷、高效,且避免了SMV侵染性克隆在大肠杆菌中的不稳定问题。SMV全长侵染性cDNA克隆的构建,为进一步研究SMV复制和发病的分子机制奠定了基础。     

国家种质库保存大豆和菜豆种质的种传病毒检测

以国家种质中期库提供的300份大豆、100份菜豆种质为材料,分别采用血清学和分子生物学方法,对种传病毒的种类进行了检测。结果表明:在大豆种质中检测出大豆花叶病毒(SMV)、黄瓜花叶病毒(CMV)、苜蓿花叶病毒(AMV)3种病毒,阳性检出率分别为25.33%(76份)、13.67%(41份)和4.67%(14份)。大豆种质中还存在大豆花叶病毒与黄瓜花叶病毒、大豆花叶病毒与苜蓿花叶病毒的复合侵染。在菜豆种质中检测出菜豆普通花叶病毒(BCMV)阳性材料92份,种质带毒率高达92%。这些信息将会对今后采取相关措施提高国家种质库保存的豆类种质的质量提供帮助。     

Mutational analysis of interaction between coat protein and helper component-proteinase of Soybean mosaic virus involved in aphid transmission

Soybean mosaic virus (SMV), a member of the genus Potyvirus , is transmitted by aphids in a non-persistent manner. It has been well documented that the helper component-proteinase (HC-Pro) plays a role as a 'bridge' between virion particles and aphid stylets in the aphid transmission of potyviruses. Several motifs, including the KITC and PTK motifs on HC-Pro and the DAG motif on the coat protein (CP), have been found to be involved in aphid transmission. Previously, we have shown strong interaction between SMV CP and HC-Pro in a yeast two-hybrid system (YTHS). In this report, we further analysed this CP–HC-Pro interaction based on YTHS and an in vivo binding assay to identify crucial amino acid residues for this interaction. Through this genetic approach, we identified two additional amino acid residues (H256 on CP and R455 on HC-Pro), as well as G12 on the DAG motif, crucial for the CP–HC-Pro interaction. We introduced mutations into the identified residues using an SMV infectious clone and showed that these mutations affected the efficiency of aphid transmission of SMV. We also investigated the involvement of the PTK and DAG motifs in the CP–HC-Pro interaction and aphid transmission of SMV. Our results support the concept that physical interaction between CP and HC-Pro is important for potyviral aphid transmission. Based on the combination of our current results with previous findings, the possibility that aphid transmission may be regulated by more complex molecular interactions than the simple involvement of HC-Pro as a bridge is discussed.     

Effect of Different Strains of Soybean Mosaic Virus on Growth, Maturity, Yield, Seed Mottling and Seed Transmission in Several Soybean Cultivars

Eighty soybeans (Glyane max Merr) cultivars, includingx a resistant line Oxley 615 were each inoculated with seven streams of soybean mosaic virus SMV. Susceptible cultivers produced smaller plants with delaved maturity, and reduced seed yield relative to the non inoculared plants Someptible cultivars had.a higher level of morrled seeds and seat transmission ot SMV from the morrled seeds than the resistance line Oxley 615. The SMV strain cultivar interaction was significant for all traits, suggsesting that soybean cultivars should be tested against specific SMV strains to determine their response to this virus.     

利用小RNA深度测序技术鉴定半夏病毒病病原

RNA沉默是真核生物体内由病毒来源的干扰小RNA（virus derived small interfering RNA, vsiRNA）沉默复合物介导目标RNA特异降解的一种保守机制,通过对vsiRNA分析可进行植物病毒病原鉴定。本文利用小RNA深度测序技术对感病半夏叶片进行鉴定,结果发现,表现典型花叶症状的半夏叶片受到大豆花叶病毒（Soybean mosaic virus, SMV）、黄瓜花叶病毒（Cucumber mosaic virus, CMV）、芋花叶病毒（Dasheen mosaic virus, DsMV）、魔芋花叶病毒（Konjac mosaic virus, KoMV）、烟草花叶病毒（Tobacco mosaic virus, TMV）等多种病毒的复合侵染。为明确SMV山西半夏分离物（SMV-SXBX）的进化关系,进行SMV-SXBX全基因组克隆与分析,获得SMV-SXBX全长为9 735 nt,编码一个由3 105个氨基酸组成的多聚蛋白质。通过核苷酸与氨基酸序列比对发现,SMV-SXBX与半夏分离物P同源性最高,分别为91.1%和94.1%,且系统发育分析表明,SMV-SXBX与半夏SMV分离物P聚为一簇。同时,也对vsiRNA进行了系统分析,研究结果有望为半夏SMV的有效防治提供一定科学依据。     

Evidence for Complementation of Plant Potyvirus Pathogenic Strains in Mixed Infection

Several strains of soybean mosaic virus (SMV) can be differentiated on the basis of the phenotypic response of various soybean cultivars (e.g., the soybean line Williams ‘82 is susceptible to all SMV strains, whereas the lines P. I. 96983, L78-379, and Davis are functionally immune to SMV strain G2 but susceptible to strains G7 and G7a). Inoculation of the immune lines with G2, followed 2 days later by inoculation with G7 or G7a, resulted in systemic spread of the avirulent SMV G2. Further evidence, suggests that complementation groups of SMV strains may exist.     

大豆花叶病毒研究进展

大豆花叶病毒（Soybean mosaic virus,SMV）病是在世界范围内广泛分布并普遍发生的病毒病害之一,导致大豆严重减产和种质衰退。这引起了国内外许多学者的科研兴趣,研究内容涉及SMV株系划分与发生分布、传播流行方式、寄主上的症状和影响因素、基因组结构组成及各基凶功能、植物生理生化抗性、大豆SMV抗性遗传育种等各方面。本文综述了近年来国内外SMV的科研方向和进展,旨在为进一步的研究提供依据。     

Robust RNAi-mediated resistance to infection of seven potyvirids in soybean expressing an intron hairpin <Emphasis Type="Italic">NIb</Emphasis> RNA

Viral pathogens, such as soybean mosaic virus (SMV), are a major constraint in soybean production and often cause significant yield loss and quality deterioration. Engineering resistance by RNAi-mediated gene silencing is a powerful strategy for controlling viral diseases. In this study, a 248-bp inverted repeat of the replicase (nuclear inclusion b, NIb) gene was isolated from the SMV SC3 strain, driven by the leaf-specific rbcS2 promoter from Phaseolus vulgaris, and introduced into soybean. The transgenic lines had significantly lower average disease indices (ranging from 2.14 to 12.35) than did the non-transformed (NT) control plants in three consecutive generations, exhibiting a stable and significantly enhanced resistance to the SMV SC3 strain under field conditions. Furthermore, seed mottling did not occur in transgenic seeds, whereas the NT plants produced ~90% mottled seeds. Virus resistance spectrum screening showed that the greenhouse-grown transgenic lines exhibited robust resistance to five SMV strains (SC3, SC7, SC15, SC18, and a recombinant SMV), bean common mosaic virus, and watermelon mosaic virus. Nevertheless, no significantly enhanced resistance to bean pod mottle virus (BPMV, Comovirus) was observed in the transgenic lines relative to their NT counterparts. Consistent with the results of resistance evaluation, the accumulation of each potyvirid (but not of BPMV) was significantly inhibited in the transgenic plants relative to the NT controls as confirmed by quantitative real-time (qRT-PCR) and double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). These results demonstrate that robust RNAi-mediated resistance to multiple potyvirids in soybean was conferred by expressing an intron hairpin SMV NIb RNA.     

Rapid Differentiation of Soybean Mosaic Virus Isolates by Antigenic Signature Analysis

Antigenic differences among 14 soybean mosaic virus (SMV) isolates were shown by signature analysis employing a panel of nine well-characterized monoclonal antibodies. Sets of binding curves were generated by reacting aliquots of serial dilutions of leaf extracts from soybean plants infected with each strain of SMV simultaneously with each antibody in the panel. An iterative alignment procedure allowed comparison of binding profiles from different assays in which the starting concentrations of SMV antigen were unknown. Desiccation of infected soybean leaf tissue altered antigenic signatures by inducing changes in reactivity of epitopes recognized by certain antibodies. The method was useful for the elucidation of subtle antigenic differences amongSMV strains. The procedure requires a minimum amount of tissue processing, the analysis is rapid and sensitive, and it requires neither purification nor knowledge of concentration of virus in ground leaf tissue from infected plants.     

Increased multiple virus resistance in transgenic soybean overexpressing the double-strand RNA-specific ribonuclease gene PAC1

Viruses constitute a major constraint to soybean production worldwide and are responsible for significant yield losses every year. Although varying degrees of resistance to specific viral strains has been identified in some soybean genetic sources, the high rate of mutation in viral genomes and mixed infections of different viruses or strains under field conditions usually hinder the effective control of viral diseases. In the present study, we generated transgenic soybean lines constitutively expressing the double-strand RNA specific ribonuclease gene PAC1 from Schizosaccharomyces pombe to evaluate their resistance responses to multiple soybean-infecting virus strains and isolates. Resistance evaluation over three consecutive years showed that the transgenic lines displayed significantly lower levels of disease severity in field conditions when challenged with soybean mosaic virus (SMV) SC3, a prevalent SMV strain in soybean-growing regions of China, compared to the non-transformed (NT) plants. After inoculation with four additional SMV strains (SC7, SC15, SC18, and SMV-R), and three isolates of bean common mosaic virus (BCMV), watermelon mosaic virus (WMV), and bean pod mottle virus (BPMV), the transgenic plants exhibited less severe symptoms and enhanced resistance to virus infections relative to NT plants. Consistent with these results, the accumulation of each virus isolate was significantly inhibited in transgenic plants as confirmed by quantitative real-time PCR and double antibody sandwich enzyme-linked immunosorbent assays. Collectively, our results showed that overexpression of PAC1 can increase multiple virus resistance in transgenic soybean, and thus provide an efficient control strategy against RNA viruses such as SMV, BCMV, WMV, and BPMV.

     

Further Studies on Mosaic Disease of Maize (Zea mays L.)

Schizaphis graminum (Rondani) is proved to be an additional vector of maize mosaic virus (MMV). The pH range for the infectivity of the virus in extracted juice is found to be from 4.4 to 9.0, the optimum being 5.6 to 7.2. Effect of certain chemicals on the virusin vitro has also been studied. Cross protection between MMV and Sugar-cane mosaic virus (SMV) indicated positive results. It has been concluded on the basis of similar physical properties, tolerance towards certain chemicals, host range, symptomatology, aphid vectors and positive immunological tests, that MMV and SMV are related viruses.     

ACC deaminase‐containing plant growth‐promoting rhizobacteria protect Papaver somniferum from downy mildew

Aims

Analysis of the stability and safety of Sulfolobus monocaudavirus 1 (SMV1) during passage through the mammalian GI tract.

Methods and Results

A major challenge of using nano‐vectors to target gut microbiome is their survival during passage through the extremely acidic and proteolytic environment of the mammalian GI tract. Here, we investigated the thermo‐acidophilic archaeal virus SMV1 as a candidate therapeutic nano‐vector for the distal mammalian GI tract microbiome. We investigated the anatomical distribution, vector stability and immunogenicity of this virus following oral ingestion in mice and compared these traits to the more classically used Inovirus vector M13KE. We found that SMV1 particles were highly stable under both simulated GI tract conditions (in vitro) and in mice (in vivo). Moreover, SMV1 could not be detected in tissues outside the GI tract and it elicited a nearly undetectable inflammatory response. Finally, we used human intestinal organoids (HIOs) to show that labelled SMV1 did not invade or otherwise perturb the human GI tract epithelium.

Conclusion

Sulfolobus monocaudavirus 1 appeared stable and safe during passage though the mammalian GI tract.

Significance and Impact of the Study

This is the first study evaluating an archaeal virus as a potential therapeutic nanoparticle delivery system and it opens new possibilities for future development of novel nanoplatforms.     

Factors affecting aphid acquisition and transmission of soybean mosaic virus

Myzus persicae transmitted soybean mosaic virus (SMV) most efficiently following 30 or 60 s acquisition probes on infected plants. There were no differences in susceptibility to SMV infection of soybean plants 1 to 12 wk old, but symptoms were more severe in plants inoculated when young than when old. Soybeans inoculated between developmental stages R3 and R6 only showed yellowish-brown blotching on one or more leaves. There were no observable differences in the time of appearance or type of symptoms shown by soybean seedlings inoculated either by sap or by aphids; infected plants became acquisition hosts for aphids 5–6 days after inoculation. There was no change in the efficiency with which M. persicae transmitted SMV from source plants up to 18 wk after inoculation. M. persicae transmitted SMV from leaves of field-grown soybeans when plants were inoculated at developmental stages V6, R2, and R3 and tested as sources 57–74 days after inoculation but not from plants inoculated at R5 and tested as sources 14 to 32 days after inoculation. M. persicae acquired SMV from soybean buds, flowers, green bean pods, and unifoliolate, trifoliolate, and senescent leaves. Middle-aged and deformed leaves were better sources of the virus than buds, unfolding and old symptomless leaves. The results are being incorporated into a computer model of SMV epidemiology.     

A tissue culture method for the preservation of Soybean mosaic virus strains

The activity and longevity of Soybean mosaic virus (SMV) in soybean callus culture were investigated with 11 SMV strains which are distinguished by differential reactions on soybean cultivars [Glycine max (L.) Merr.]. Dual cultures (soybean callus and SMV) were initiated by direct culture of SMV-infected leaves from susceptible soybean plants on Msoy and MS agar medium. Established SMV-callus cultures were maintained at 25 °C under light, subcultured to fresh MS medium at 2-month intervals or as necessary, and assayed periodically for virus infectivity. The infected calluses on MS medium grew better and stayed active longer than those on Msoy medium. At 10–15 °C, calluses and SMV were viable and active for 13–15 weeks or longer without subculture. The infectivity of SMV from callus cultures was comparable with that of SMV from infected plants, and remained stable for more than a year through five successive subcultures. Callus tissues of dual cultures were uniformly infected by SMV, thus ensuring infectious subcultures by random transfers. Production of in vitro inoculum can be significantly increased by multiple subcultures. Biological integrity of the SMV cultures was maintained with no change of viral virulence and pathotype. The method is of value for preserving a collection of SMV strains in a highly infectious and readily available form and reduces the chance of contamination or loss in viability.     

Soybean leaf pubescence affects aphid vector transmission and field spread of soybean mosaic virus

A study was undertaken to determine the influence of trichome density on the spread of non-persistently transmitted plant viruses by aphid vectors. A system using soybean plants and soybean mosaic virus (SMV) tested the hypothesis that greater leaf trichome density inhibits probing activity of vector species, leading to reduced virus spread and retarded virus epidemics under field conditions. Probing activity of three important aphid vectors of SMV, Myzus persicae, Rhopalosiphum maidis, and Aphis citricola, was affected by the density of soybean leaf trichomes. Less pubescent and glabrous isolines elicited greater probing activity than did densely pubescent isolines. Among the parameters considered, probe duration was found to be species specific, whereas the following traits were consistent among species for the denser isolines: reduced numbers of probes, greater length of time to first probe, and less time spent probing. Laboratory transmission of soybean mosaic virus was reduced in the more densely pubescent isolines by the vector species tested. Field spread of SMV was negatively correlated with density of pubescence. In our system, we found that denser leaf pubescence retards field epidemics of non-persistently transmitted plant viruses.     

Molecular Characterization of a Chinese Soybean Mosaic Virus Isolate by RT-PCR,cDNA Sequence Analysis and Direct Expression of PCR Products in Bacteria

Soybean mosaic virus (SMV) causes one of the most severe viral diseases in soybean ( Glycine max L. ) and is known to contain many pathogenically and serologically related isolates. In the present study, the authors have obtained cDNAs to all cistrons of a Chinese SMV isolate, SMV-ZK, by RT-PCR. By analyzing the nucleotide and amino acid sequence of the HC-PRO, Nib and CP cistrons, it was found that SMV-ZK was highly homologous to the G2 strain of SMV, thus confirming the existence of G2-1ike isolates in soybean crop in China. The amplified cDNAs were directly cloned into a bacterial expression vector. With the exception of the P3 cistron, expression of the cDNAs of all other cistrons in bacteria gave rise to polypeptides of expected molecular weight. The expressed viral proteins were subsequently purified by gel elution. The preparation of viral-specific cDNAs and gene products will be useful in future functional study of the SMV genome.     

Inheritance and allelism of resistance to soybean mosaic virus in Zao18 soybean from China

Soybean mosaic disease caused by soybean mosaic virus (SMV) occurs wherever soybean [Glycine max (L.) Merr.] is grown and is considered one of the most important soybean diseases in many areas of the world. Use of soybean cultivars with resistance to SMV is a very effective way of controlling the disease. China has rich soybean germplasm, but there is very limited information on genetics of SMV resistance in Chinese soybean germplasm and reaction of the resistance genes to SMV strains G1-G7. There also is no report on allelic relationships of resistance genes in Chinese soybeans with other named genes at the three identified loci Rsv1, Rsv3, and Rsv4. The objectives of this study were to examine reactions of Chinese soybean cultivar Zao18 to SMV strains G1-G3 and G5-G7, to reveal the inheritance of SMV resistance in Zao18 and to determine the allelic relationship of resistance genes in Zao18 with previously reported resistance genes. Zao18 was crossed with the SMV-susceptible cultivar Lee 68 to study the inheritance of resistance. Zao18 was also crossed with the resistant lines PI96983, L29, and V94-5152, which possess Rsv1, Rsv3, and Rsv4, respectively, to examine the allelic relationship between the genes in Zao18 and genes at these three loci. Our research results indicated that Zao18 possesses two independent dominant genes for SMV resistance, one of which is allelic to the Rsv3 locus; the other is allelic with Rsv1. The presence of both genes (Rsv1 and Rsv3) in Zao18 confers resistance to SMV strains G1-G7.