Soybean RNA interference lines silenced for eIF4E show broad potyvirus resistance

Soybean mosaic virus (SMV), a potyvirus, is the most prevalent and destructive viral pathogen in soybean-planting regions of China. Moreover, other potyviruses, including bean common mosaic virus (BCMV) and watermelon mosaic virus (WMV), also threaten soybean farming. The eukaryotic translation initiation factor 4E (eIF4E) plays a critical role in controlling resistance/susceptibility to potyviruses in plants. In the present study, much higher SMV-induced eIF4E1 expression levels were detected in a susceptible soybean cultivar when compared with a resistant cultivar, suggesting the involvement of eIF4E1 in the response to SMV by the susceptible cultivar. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that soybean eIF4E1 interacted with SMV VPg in the nucleus and with SMV NIa-Pro/NIb in the cytoplasm, revealing the involvement of VPg, NIa-Pro, and NIb in SMV infection and multiplication. Furthermore, transgenic soybeans silenced for eIF4E were produced using an RNA interference approach. Through monitoring for viral symptoms and viral titers, robust and broad-spectrum resistance was confirmed against five SMV strains (SC3/7/15/18 and SMV-R), BCMV, and WMV in the transgenic plants. Our findings represent fresh insights for investigating the mechanism underlying eIF4E-mediated resistance in soybean and also suggest an effective alternative for breeding soybean with broad-spectrum viral resistance.     

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.     

Genetics of resistance to two strains of soybean mosaic virus in differential soybean genotypes

There are seven pathotypes of soybean mosaic virus (SMV) representing seven strain groups (G1-G7) in the United States. Soybean genotypes [Glycine max (L.) Merr.] may exhibit resistant (R), susceptible (S), or necrotic (N) reactions upon interacting with different SMV strains. This research was conducted to investigate whether reactions to two SMV strains are controlled by the same gene or by separate genes. Two SMV-resistant soybean lines, LR1 and LR2, were crossed with the susceptible cultivar Lee 68. LR1 contains a resistance gene Rsv1-s and is resistant to strains G1-G4 and G7. LR2 contains the Rsv4 gene and is resistant to strains G1-G7. Two hundred F(2:3) lines from LR1 x Lee 68 and 262 F(2:3) lines from LR2 x Lee 68 were screened for SMV reaction. Seeds from each F2 plant were randomly divided into two subsamples. A minimum of 20 seeds from each subsample were planted in the greenhouse and plants were inoculated with either G1 or G7. G1 is the least virulent, whereas G7 is the most virulent strain of SMV. The results showed that all the F(2:3) lines from both crosses exhibited the same reaction to G1 and G7. No recombinants were found in all the progenies for reactions to G1 and G7 in either cross. The results indicate that reactions to both G1 and G7 are controlled by either the same gene or very closely linked genes. This research finding is valuable for studying the resistance mechanism and interactions of soybean genotypes and SMV strains and for breeding SMV resistance to multiple strains.     

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。     

抗除草剂转基因大豆后代遗传分析

分析了来源于农杆菌介导的4个独立的大豆转化系的后代遗传特性。分别采用种子切片GUS染色方法和除草剂涂抹以及喷洒方法检测gus报告基因和抗除草剂bar基因在后代的表达。其中3个转化系T1代gus基因和bar基因能够以孟德尔方式3：1连锁遗传,说明这2个基因整合在大豆基因组的同一位点。这3个转化系在T2代获得了纯合的转化系,并能够稳定遗传至T5代。有一个转化系在T1代GUS和抗除草剂检测都为阴性,但通过Southern杂交证明转基因存在于后代基因组,显示发生了转基因沉默。为了证明转基因沉默是转录水平还是转录后水平,T1代植物叶片接种大豆花叶病毒（SMV）并不能抑制转基因沉默,说明该转化系基因沉默可能不是发生在转录后水平。     

A <Emphasis Type="Italic">bean common mosaic virus</Emphasis> (BCMV)-resistance gene is fine-mapped to the same region as <Emphasis Type="Italic">Rsv1</Emphasis>-<Emphasis Type="Italic">h</Emphasis> in the soybean cultivar Suweon 97

Key message

In the soybean cultivar Suweon 97, BCMV-resistance gene was fine-mapped to a 58.1-kb region co-localizing with the Soybean mosaic virus (SMV)-resistance gene, Rsv1-h raising a possibility that the same gene is utilized against both viral pathogens.

Abstract

Certain soybean cultivars exhibit resistance against soybean mosaic virus (SMV) or bean common mosaic virus (BCMV). Although several SMV-resistance loci have been reported, the understanding of the mechanism underlying BCMV resistance in soybean is limited. Here, by crossing a resistant cultivar Suweon 97 with a susceptible cultivar Williams 82 and inoculating 220 F₂ individuals with a BCMV strain (HZZB011), we observed a 3:1 (resistant/susceptible) segregation ratio, suggesting that Suweon 97 possesses a single dominant resistance gene against BCMV. By performing bulked segregant analysis with 186 polymorphic simple sequence repeat (SSR) markers across the genome, the resistance gene was determined to be linked with marker BARSOYSSR_13_1109. Examining the genotypes of nearby SSR markers on all 220 F₂ individuals then narrowed down the gene between markers BARSOYSSR_13_1109 and BARSOYSSR_13_1122. Furthermore, 14 previously established F_2:3 lines showing crossovers between the two markers were assayed for their phenotypes upon BCMV inoculation. By developing six more SNP (single nucleotide polymorphism) markers, the resistance gene was finally delimited to a 58.1-kb interval flanked by BARSOYSSR_13_1114 and SNP-49. Five genes were annotated in this interval of the Williams 82 genome, including a characteristic coiled-coil nucleotide-binding site-leucine-rich repeat (CC-NBS-LRR, CNL)-type of resistance gene, Glyma13g184800. Coincidentally, the SMV-resistance allele Rsv1-h was previously mapped to almost the same region, thereby suggesting that soybean Suweon 97 likely relies on the same CNL-type R gene to resist both viral pathogens.

     

獐茅HAK基因表达调控区的分离及其在水稻中的功能分析

獐茅高亲和性K+转运蛋白基因(AlHAK1)是从单子叶禾本科盐生植物獐茅(Aeluropus littoralis(Gouan)Parl)中克隆,对于细胞营养和离子渗透调节起关键作用。为了进一步了解AlHAK1基因的表达调控机制,文章采用基因组步移法分离了AlHAK1基因转录起始位点上游长度约1.3 kb的启动子区域。启动子顺式元件分析显示该序列具有典型的TATA和CAAT盒,以及一些与植物生长发育和环境响应相关的顺式元件。为了明确AlHAK1启动子的功能,将其与GUS基因融合构建到植物表达载体pCAMBIA1301上,通过农杆菌介导转化法导入水稻中。对转基因植株进行GUS组织化学染色,结果显示在转化AlHAK1启动子水稻的根、茎、叶、花药和内外稃部位均检测到GUS活性。GUS荧光定量分析显示AlHAK1启动子调节GUS表达活性低于组成型启动子CaMV35S和Ubiquitin,但其根部和茎部的GUS活性相对较高。对转化植株进行不同胁迫处理后检测GUS活性,结果表明受到ABA、干旱、高温的诱导后其茎部和根部GUS活性有所提高,推测位于该启动子-682 bp的HSE元件和-1 268 bp的MybBS元件可能在高温、ABA和干旱诱导的表达调控中起作用。     

大豆种质对SMV成株和种粒斑驳抗性的SSR标记辅助鉴定

对186份大豆种质资源进行了成株和种粒斑驳抗性鉴定,并利用与成株抗性及种粒斑驳抗性分别相关的SSR标记验证抗病毒分子辅助选择的可行性。结果表明：接种SMV1,选出成株和种粒双抗种质38份,成株抗病种质149份,种粒抗病种质45份,成株和种粒双感种质26份。利用与成株抗性相关的SSR标记Sat_229、Sat_317、Satt335、Satt160、Satt516、Sat_309进行检测,抗病毒资源筛选的准确率分别达到68．9％、74．3％、71．1％、69．8％、77．4％和68．2％。利用与种粒斑驳抗性相关的SSR标记Sat_297、Sat_229、Sat_317、Satt335、Set_188、Satt160、Satt516、Sat_133进行检测,Sat_317标记准确率达79．1％,标记Sat_229、Satt335、Satt516和Sat_133抗病毒资源筛选的准确率均达70％以上,可以用作抗病毒分子辅助育种的选择标记。     

西瓜果实特异性基因wml1的5‘端上游调控序列的分离

西瓜（Citrullus vulgris Schrad.)ADP-葡萄糖焦磷酸化酶大亚基基因wml1的表达具有果实特异性。本文利用Uneven PCR技术成功地从西瓜基因组DNA中分离出一段长1864bp、位于wml1基因5‘端上游的新序列。该序列含有TATA盒和CAAT盒,具典型的启动子特征。克隆序列中180bp-1752bp和958bp-1752bp两个片段分别与GUS基因融合进行瞬间表达试验,结果初步表明180bp-1752bp片段具有果实特异性启动子活性,转录调控元件位于序列的180bp-958bp。     

Complementary action of two independent dominant genes in Columbia soybean for resistance to Soybean mosaic virus

A stem-tip necrosis disease was observed in the soybean [Glycine max (L.) Merr.] cultivar Columbia and its derivative OX686 when infected with a necrosis-causing strain of Soybean mosaic virus (SMV) in Canada. A dominant gene named Rsv3 was found in OX686 for the necrotic reaction. In the present research we have found that Columbia is resistant to all known SMV strains G1-G7, except G4. Genetic studies were conducted to investigate the inheritance of resistance in Columbia and interactions of resistance gene(s) with SMV strains. Columbia was crossed with a susceptible cultivar, Lee 68, and with resistant lines PI96983, Ogden, and LR1, each possessing a resistance gene at the Rsv1 locus. F(1) individuals, F(2) populations, and F(2:3) lines from these crosses were inoculated with G7 or G1 in the greenhouse. Our inheritance data confirmed the presence of two independent dominant genes for SMV resistance in Columbia. Results from allelism tests further demonstrate that the two genes (referred to as R3 and R4 in this article) in Columbia were independent of the Rsv1 locus. R3 appears to be the same gene previously reported as Rsv3 in OX686, which was derived from Columbia. The R3 gene confers resistance to G7, but necrosis to G1. The other gene, R4, conditions resistance to G1 and G7 at the early seedling stage and then a delayed mild mosaic reaction (late susceptible) 3 weeks later. Plants carrying both the R3 and R4 genes were completely resistant to both G1 and G7, indicating that the two genes interact in a complementary fashion. Plants heterozygous for R3 or R4 exhibited systemic necrosis or late susceptibility, suggesting that the resistance is allele dosage dependent. The R4 gene appeared epistatic to R3 since it masked expression of necrosis associated with the response of R3. The complementary interaction of two resistance genes, as exhibited in Columbia, can be useful in development of soybean cultivars with multiple and durable resistance to SMV.     

Genetic analysis of resistance to soybean mosaic virus in j05 soybean

Soybean cultivar J05 was identified to be resistant to the most virulent strain of soybean mosaic virus (SMV) in northeastern China. However, the reaction of J05 to SMV strains in the United States of America is unknown, and genetic information is needed to utilize this germplasm in a breeding program. The objectives of this study were to determine the reaction of J05 to all US strains of SMV (G1-G7), the inheritance of SMV resistance in J05, and the allelic relationship of resistance genes in J05 with other reported resistance genes. J05 was crossed with susceptible cultivar Essex (rsv) to study the inheritance of SMV resistance. J05 was also crossed with PI 96983 (Rsv1), L29 (Rsv3), and V94-5152 (Rsv4) to test the allelism of resistance genes. F(2) populations and F(2:3) lines from these crosses were inoculated with G1 or G7 in the greenhouse. Inheritance and allelism studies indicate that J05 possesses 2 independent dominant genes for SMV resistance, one at the Rsv1 locus conferring resistance to G1 and necrosis to G7 and the other at the Rsv3 locus conditioning resistance to G7 but susceptibility to G1. The presence of both genes in J05 provides resistance to G1 and G7. J05 is unique from the previous sources that carry 2 genes of Rsv1Rsv3 and will be useful in breeding for SMV resistance.     

<Emphasis Type="Italic">Organogenic callus</Emphasis> as the target for plant regeneration and transformation via <Emphasis Type="Italic">Agrobacterium</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.)

A regeneration and transformation system has been developed using organogenic calluses derived from soybean axillary nodes as the starting explants. Leaf-node or cotyledonary-node explants were prepared from 7 to 8-d-old seedlings. Callus was induced on medium containing either Murashige and Skoog (MS) salts or modified Finer and Nagasawa (FNL) salts and B5 vitamins with various concentrations of benzylamino purine (BA) and thidiazuron (TDZ). The combination of BA and TDZ had a synergistic effect on callus induction. Shoot differentiation from the callus occurred once the callus was transferred to medium containing a low concentration of BA. Subsequently, shoots were elongated on medium containing indole-3-acetic acid (IAA), zeatin riboside, and gibberellic acid (GA). Plant regeneration from callus occurred 90 ∼ 120 d after the callus was cultured on shoot induction medium. Both the primary callus and the proliferated callus were used as explants for Agrobacterium-mediated transformation. The calluses were inoculated with A. tumefaciens harboring a binary vector with the bar gene as the selectable marker gene and the gusINT gene for GUS expression. Usually 60–100% of the callus showed transient GUS expression 5 d after inoculation. Infected calluses were then selected on media amended with various concentrations of glufosinate. Transgenic soybean plants have been regenerated and established in the greenhouse. GUS expression was exhibited in various tissues and plant organs, including leaf, stem, and roots. Southern and T₁ plant segregation analysis of transgenic events showed that transgenes were integrated into the soybean genome with a copy number ranging from 1–5 copies.     

Interactive effects of aphid feeding and virus infection on host gene expression and volatile compounds in salt-stressed soybean, <Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.

Saline soils are becoming an important limiting factor in production agriculture. Soybean cultivars [Glycine max (L.) Merr.] differ in their ability to tolerate salt stress with those that cannot limit ion uptake into leaves being salt sensitive. Those that can partially limit ion uptake into leaves are generally more salt tolerant. Soybean mosaic virus (SMV) is an important viral pathogen of soybean worldwide and is commonly transmitted by the soybean aphid, Aphis glycines Matsumura. In this study, we investigate the interaction of salt stress in soybean with SMV infection and infestation by the soybean aphid by measuring aphid populations in a no-choice assay, gene expression levels, and the induction of volatile organic compounds using static headspace GC–MS analysis. Salt stress and SMV infection both reduced total aphid populations, though SMV did not reduce the total number of aphids per gram of fresh weight. Aphid suppression of a calcium EF hand gene and OPR1 was lost when salt-sensitive soybean plants were salt stressed and when salt-tolerant plants were subjected to all three stressors. The relative levels of SMV in aphid-infested soybeans were increased by salt stress in the salt-sensitive cultivar, whereas SMV levels decreased in the salt-tolerant cultivar. Static headspace collection of volatile organic compounds revealed that salt stress and SMV infection had suppressive activities on aphid-induced terpenes. These results suggest that although salt stress has a negative impact on aphid population size, the changes in volatiles and SMV levels could alter the incidence of SMV in salt-stressed fields.     

Analysis of probenazole-responsiveness of rice RPR1 upstream fragments.

RPR1 (rice probenazole-responsive) is a rice gene, the expression of which is responsive to probenazole (PBZ), a synthetic compound that may act as a plant defense activator. It has been shown that RPR1 gene may be involved in disease resistance responses. In this study, a series of amplified fragments from the rice RPR1 promoter region, including 2,416 bp, 1,574 bp, 819 bp, 568 bp and 208 bp fragments upstream to the ATG translation start site, were prepared and linked to the coding region of beta-glucuronidase (GUS) gene. Analysis of GUS gene transient expression in rice calli demonstrated that the 568 bp fragment was sufficient for probenazole responsiveness. Analysis of GUS gene stable expression in Arabidopsis thaliana indicated that the 2,416 bp and 1,574 bp fragments drove GUS expression only in shoot apical meristem and petiole. Identification of these PBZ-responsive fragments provides a basis on which PBZ-inducible gene regulatory systems can be constructed for experimental analysis of gene expression and for field application.     

Partitioning two- and three-trophic-level effects of resistant plants on the predator, Nabis roseipennis

Two- and three-trophic-level effects of resistant soybean plants on the development and fecundity of the predator Nabis roseipennis Reuter (Hemiptera: Nabidae) were determined. The nabids, like many hemipteran predators, feed on plants as well as arthropod prey. The life history traits of N. roseipennis when feeding on Pseudoplusia includens (Hübner) (Lepidoptera: Noctuidae) fed the resistant soybean cultivar PI 229358 or the susceptible Cobb' and while exposed to these resistant and susceptible soybean cultivars or a no-plant control were determined. Nymphal survival of N. roseipennis was unaffected by either prey or plant treatment. Development time was extended and fecundity of N. roseipennis was reduced by feeding on prey fed the resistant plant but not by exposure to resistant soybean plants. Thus, three-trophic-level effects of the resistant cultivar PI 229358 on prey quality were more important than two-trophic-level effects of direct plant feeding on development and fecundity of N. roseipennis. Consistency of these results with those of other predatory hemipterans and the potential impact of diet composition on the effects of resistant plants are discussed.