Identification of Replication Specificity Determinants in Two Strains of Tomato Leaf Curl Virus from New Delhi

We used two strains of tomato leaf curl virus from New Delhi to investigate specificity in replication of their cognate genomes. The strains share 94% sequence identity and are referred to as severe and mild on the basis of symptoms on tomato and tobacco. Replication assays in tobacco protoplasts and plants showed that a single amino acid change, Asn10 to Asp in the N terminus of Rep protein, determines specificity for replication of the two strains based upon its interaction with the origin of replication (ori) sequences. The change of Asp10 to Asn in Rep protein of the mild strain coupled with point mutations at the 3rd and 10th nucleotides of the 13-mer binding site altered its replication ability, resulting in increased levels of virus accumulation. Similarly, changing Asn10 to Asp in Rep protein of the severe strain impaired replication of the virus and altered its severe phenotype in plants. Site-directed mutations made in ori and Asn10 of Rep protein suggested that Asn10 recognizes the third base pair of the putative binding site sequence GGTGTCGGAGTC in the severe strain.     

Molecular evidence of occurrence of Tomato Leaf Curl New Delhi Virus infecting cucurbits in several states in India

Abstract

Tomato Leaf Curl New Delhi Virus (ToLCNDV) is an important begomovirus constraint to the production of cucurbitaceous and other crops in India and its subcontinent. In this study first time, symptomatic samples of different cucurbits were collected from Telangana, Uttar Pradesh (Varanasi) and Madhya Pradesh location in India. The symptomatic samples were associated with begomovirus infection was confirmed by PCR amplification using specific universal primers Deng 540/541. Thirteen out of fifteen samples were amplified with newly designed bipartite specific primers JKR 58?F and JKR 59?R. In eleven out of thirteen bipartite samples contain betasatellite, confirmed by PCR amplification using specific primer CLB36F and CLB37R. The amplified PCR product with JKR58F and 59R of bitter gourd sample collected from Telangana was sequenced. The sequence was share 97.53% similarity with ToLCNDV infecting bottle gourd in Haryana in India (FN645905). Phylogenic analysis revealed that ToLCNDV infecting cucurbits are different, from ToLCNDV infecting tomato.     

Within-Host Dynamics of the Emergence of Tomato Yellow Leaf Curl Virus Recombinants

Tomato yellow leaf curl virus (TYLCV) is a highly damaging begomovirus native to the Middle East. TYLCV has recently spread worldwide, recombining with other begomoviruses. Recent analysis of mixed infections between TYLCV and Tomato leaf curl Comoros begomovirus (ToLCKMV) has shown that, although natural selection preserves certain co-evolved intra-genomic interactions, numerous and diverse recombinants are produced at 120 days post-inoculation (dpi), and recombinant populations from different tomato plants are very divergent. Here, we investigate the population dynamics that lead to such patterns in tomato plants co-infected with TYLCV and ToLCKMV either by agro-inoculation or using the natural whitefly vector Bemisia tabaci. We monitored the frequency of parental and recombinant genotypes independently in 35 plants between 18 and 330 dpi and identified 177 recombinants isolated at different times. Recombinants were detected from 18 dpi and their frequency increased over time to reach about 50% at 150 dpi regardless of the inoculation method. The distribution of breakpoints detected on 96 fully sequenced recombinants was consistent with a continuous generation of new recombinants as well as random and deterministic effects in their maintenance. A severe population bottleneck of around 10 genomes was estimated during early systemic infection–a phenomenon that could account partially for the heterogeneity in recombinant patterns observed among plants. The detection of the same recombinant genome in six of the thirteen plants analysed beyond 30 dpi supported the influence of selection on observed recombination patterns. Moreover, a highly virulent recombinant genotype dominating virus populations within one plant has, apparently, the potential to be maintained in the natural population according to its infectivity, within-host accumulation, and transmission efficiency - all of which were similar or intermediate to those of the parent genotypes. Our results anticipate the outcomes of natural encounters between TYLCV and ToLCKMV.     

Tomato Yellow Leaf Curl Virus Infection in a Monocotyledonous Weed (Eleusine indica)

Tomato yellow leaf curl virus (TYLCV) is one of the most important plant viruses belonging to the genus Begomovirus of the family Geminiviridae. To identify natural weed hosts that could act as reservoirs of TYLCV, 100 samples were collected at a TYLCV-affected tomato farm in Iksan from 2013 to 2014. The sample weeds were identified as belonging to 40 species from 18 families. TYLCV was detected in 57 samples belonging to 28 species through polymerase chain reaction using root samples including five species (Eleusine indica, Digitaria ciliaris, Echinochloa crus-galli, Panicum dichotomiflorum, and Setaria faberi) from the family Poaceae. Whitefly Bemisia tabaci-mediated TYLCV transmission from TYLCV-infected E. indica plants to healthy tomatoes was confirmed, and inoculated tomatoes showed typical symptoms, such as leaf curling and yellowing. In addition, TYLCV was detected in leaf and root samples of E. indica plants inoculated by both whitefly-mediated transmission using TYLCV-viruliferous whitefly and agro-inoculation using a TYLCV infectious clone. The majority of mastreviruses infect monocotyledonous plants, but there have also been reports of mastreviruses that can infect dicotyledonous plants, such as the chickpea chlorotic dwarf virus. No exception was reported among begomoviruses known as infecting dicots only. This is the first report of TYLCV as a member of the genus Begomovirus infecting monocotyledonous plants.     

Infectivity of Tomato Leaf Curl Hainan Virus in Tomato and Tobacco in China

Tomato leaf curl Hainan virus (ToLCHnV) was previously reported as a distinct begomovirus infecting tomato in Hainan, China. To investigate the infectivity of ToLCHnV, an infectious clone of ToLCHnV‐[CN: HaNHK7] was constructed and agro‐inoculated into Solanum lycopersicum, Nicotiana benthamiana, Nicotiana glutinosa, Petunia hybrida, Cucumis sativus, Solanum melongena and Capsicum annuum plants; it induced severe leaf curling and crinkling symptoms in these plant species except C. sativus, S. melongena and C. annuum. The induced symptoms were compared with those induced by Papaya leaf curl China virus.     

番茄抗黄化曲叶病毒育种研究进展

本文分别对近年番茄抗黄化曲叶病毒的传统育种、分子辅助育种、基因工程育种进展进行了综述.栽培番茄均不抗番茄黄化曲叶病毒,所以传统育种采用从野生近缘种中筛选抗性材料,以其为亲本与栽培番茄进行杂交来获得抗性;野生近缘种中的抗性位点Ty-1、Ty-2和Ty-3及一些QTLs先后被定位,也筛选出了可鉴定Ty-1基因的SSR-47标记及鉴定Ty-3的SCAR标记;通过转基因技术获得抗性是研究热点之一,目前转入番茄后表现出抗性的序列有TYLCV病毒的CP基因、尺EP基因的部分序列或反义序列、不编码的保守序列以及源于白粉虱的GroEL基因.同时讨论了今后的主要发展方向.     

云南番茄曲叶病是由烟草曲茎病毒引起的

从云南省德宏田间表现曲叶症状的番茄植株上分离到病毒分离物Y41,采集的带病植株在实验室可经烟粉虱(Bemisia tabaci)传播到健康的番茄.用针对非洲木薯花叶病毒(ACMV)、印度木薯花叶病毒(ICMV)及秋葵曲叶病毒(OLCV)的15种单抗对病样进行TAS-ELISA检测,结果表明,番茄曲叶病是由菜豆金色花叶病毒属(Begomovirus)病毒引起的,但其抗原表位型与我国广西报道的中国番茄黄化曲叶病毒(TYLCCV)不同.对Y41进行DNA-A全序列测定和分析表明,Y41 DNA-A全长2743个核苷酸,共编码6个ORF,其中病毒链编码AV1和AV2两个ORF,互补链编码AC1、AC2、AC3和AC4 4个ORF.对Y41及其它双生病毒CP进行同源性比较及系统进化关系分析表明,Y41属于"旧世界"的粉虱传双生病毒,与我国报道的烟草曲茎病毒(TCSV)及印度报道的番茄曲叶Karnataka病毒(ToLCKV)同源性最高,达到98.8%.进一步比较基因组发现,Y41与TCSV AV1、AV2、AC1、AC2、AC3、AC4各ORF同源性分别为98.8%、96.6%、86.4%、93.3%、89.6%和89.7%,基因间隔区(IR)、DNA-A同源性分别为92.1%和93.4%,且在基因间隔区内含有相似的重复子序列及排列方式.这些结果表明:Y41是TCSV在自然条件下侵染番茄的一个分离物.     

番茄黄化曲叶病毒对番茄叶片光合特性和叶绿体超微结构的影响

以番茄为试验材料,研究番茄黄化曲叶病毒(TYLCV)侵染对植株叶片叶绿素含量、净光合速率、气孔导度、胞间CO2浓度和叶绿体超微结构的影响.结果表明:TYLCV侵染番茄后,叶片叶绿素a、b以及总叶绿素含量分别下降50.2%、24.19%和43.84%,叶片净光合速率和气孔导度分别下降43.28%、27.07%,胞间CO2浓度增加13.04%.与健康叶片相比,叶绿体变形,叶绿体基质片层大部分消解,基粒结构消失,叶绿体外膜和内膜剥离,质壁分离和细胞膜内陷,细胞器消解.研究表明,TYLCV侵染破坏了番茄叶片的叶绿体结构,严重影响番茄叶片的光合作用.     

Recruitment of the Host Plant Heat Shock Protein 70 by Tomato Yellow Leaf Curl Virus Coat Protein Is Required for Virus Infection

A functional capsid protein (CP) is essential for host plant infection and insect transmission of Tomato yellow leaf curl virus (TYLCV) and other monopartite begomoviruses. We have previously shown that TYLCV CP specifically interacts with the heat shock protein 70 (HSP70) of the virus insect vector, Bemisia tabaci. Here we demonstrate that during the development of tomato plant infection with TYLCV, a significant amount of HSP70 shifts from a soluble form into insoluble aggregates. CP and HSP70 co-localize in these aggregates, first in the cytoplasm, then in the nucleus of cells associated with the vascular system. CP-HSP70 interaction was demonstrated by co-immunopreciptation in cytoplasmic - but not in nuclear extracts from leaf and stem. Inhibition of HSP70 expression by quercetin caused a decrease in the amount of nuclear CP aggregates and a re-localization of a GFP-CP fusion protein from the nucleus to the cytoplasm. HSP70 inactivation resulted in a decrease of TYLCV DNA levels, demonstrating the role of HSP70 in TYLCV multiplication in planta. The current study reveals for the first time the involvement of plant HSP70 in TYLCV CP intracellular movement. As described earlier, nuclear aggregates contained TYLCV DNA-CP complexes and infectious virions. Showing that HSP70 localizes in these large nuclear aggregates infers that these structures operate as nuclear virus factories.     

Papaya Leaf Curl Guangdong Virus and Ageratum Yellow Vein Virus Associated with Leaf Curl Disease of Tobacco in China

Two samples (YC7, YC27) of Nicotiana tabacum showing leaf curling, vein swelling and enations on undersides of leaves were collected in the Fujian Province of China in 2007. Virus isolates YC7‐1 and YC7‐2 (associated with betasatellite, YC7‐2β) were detected in both samples. The complete DNA‐A sequence of YC7‐1 (FJ869907) comprised 2741 nucleotides (nt). The complete DNA‐A (FJ869908) and betasatellite (FJ869909) sequence of YC7‐2 consisted of 2754 and 1344 nt, respectively. YC7‐1 had the highest nucleotide sequence identity (97.3%) with Papaya leaf curl Guangdong virus (PaLCuGuV‐[CN:Gd2:02], AJ558122). YC7‐2 had the highest sequence identity (90.1%) with Ageratum yellow vein virus (AYVV‐TW[TW:Tai:99], AF307861) and its betasatellite (96.5%) with Ageratum yellow vein betasatellite (AYVB‐[TW:CHu:02], AJ542495). These indicate that YC7‐1 and YC7‐2 are isolates of PaLCuGuV and AYVV, respectively. Symptoms including leaf curling, vein swelling and enations on undersides of leaves were observed in N. tabacum and N. glutinosa when infected by whiteflies with sample YC7 as the viral source under greenhouse conditions. PCR results showed that these infected plants contained both YC7‐1 and YC7‐2/YC7‐2β. To our knowledge, this is the first report of PaLCuGuV and AYVV/AYVB co‐infecting N. tabacum in China.     

Begomoviruses Associated with Yellow Leaf Curl Disease of Tomato in Iran*

The occurrence of Tomato yellow leaf curl virus (TYLCV; genus Begomovirus, family Geminiviridae) in the major tomato‐growing areas of Iran was determined using TAS‐ELISA and PCR. The nucleotide sequences of the coat protein (CP) gene and intergenic region (IR) of eight Iranian isolates were determined. CP nucleotide identities among the Iranian isolates were 96–98%, and showed 94–96% identity with TYLCV‐IR [IR:Ira:98] and TYLCV‐IL [IL:Reo:86]. However, they showed low identity (68–69%) with ToLCIRV‐[IR:Ira]. Sequence analyses of IR indicated that seven Iranian isolates had sequence identity of 93–100% with each other, and 76% identity with the Jiroft isolate; identities of 75–79% with TYLCV‐IR[IR:Ira:98] were observed in every case, and 59–62% identity with ToLCIRV‐[IR:Ira]. The IR nucleotide sequences of Iranian isolates showed 92–93% identity with TYLCV‐IL[IL:Reo:86], except the Jiroft isolate (75%). The CP and IR sequence analyses suggested that eight Iranian TYLCV isolates probably differ from ToLCIRV‐[IR:Ira]. Based on IR sequence comparisons and phylogenetic analyses, the Iranian isolates were divided into two groups. The first major group (A), consists of seven virus isolates, was most closely related to TYLCV‐IL[IL:Reo:86], and relatively divergent from TYLCV‐IR [IR:Ira:98] and ToLCIRV‐[IR:Ira]. However, the Jiroft isolate from group B did not show high similarity with TYLCV‐IR[IR:Ira:98], ToLCIRV‐[IR:Ira], and TYLCV‐IL[IL:Reo:86], suggesting that the isolate may be a divergent variant. The differences are in a range that suggests different strains or species from TYLCV‐IR[IR:Ira:98] and ToLCIRV‐[IR:Ira] are probably associated with tomato yellow leaf curl disease in Iran.     

Begomoviruses Associated with Leaf Curl Disease of Tomato in Java,Indonesia*

We report that several begomoviruses are associated with tomato leaf curl disease in Java, Indonesia. Tomato plants with leaf curl symptoms were collected from Bandung (west Java), Purwokerto (central Java), Magelang (central Java) and Malang (east Java) of Indonesia, the major tomato‐growing areas of the country. Viruses were detected using the polymerase chain reaction (PCR), with universal primers for the genus Begomovirus. PCR‐amplified fragments were cloned and sequenced. Based on sequence comparisons and phylogenetic analyses, the viruses were divided into three groups. With respect to amino acid (aa) identities of the N‐terminal halves of the coat proteins compared in this study, group I was most closely related to Ageratum yellow vein virus (AYVV) (97%), Ageratum yellow vein China virus‐[Hn2] (AYVCNV‐[Hn2]) (96%) and Ageratum yellow vein virus‐[Taiwan] (AYVV‐[Tai]) (95%), and ageratum‐infecting begomovirus from Java (99%). Group II had high sequence identity with a tentative species of tomato leaf curl Java virus (ToLCJAV) (96% aa) for the CP. Group III was most closely related to a proposed species of Pepper yellow leaf curl Indonesia virus (PepYLCIDV) (90% aa identity) by its partial CP sequence.     

PCR Based Diagnosis of Begomoviruses Associated with Tomato Leaf Curl Disease in India

This paper reports development of a set of common primers and four sets of virus specific primers to detect ToLCNDV, ToLCBV, ToLCGV and ToLCKV, which are associated with tomato leaf curl disease (TomLCD) in India. The primer sets were first validated by amplification of desired genomic fragments from the cloned genomes of the four begomoviruses and then were tested for the detection of these viruses in field samples collected from different locations in India. Of the 26 TomLCD samples tested by PCR, 22 tested positive with the set of common primers and 16 tested positive for the target begomoviruses. ToLCNDV was detected in four samples, ToLCBV in six, ToLCKV in four, and ToLCGV in five samples. This limited survey shows that ToLCNDV and ToLCBV are distributed in northern and southern India, whereas ToLCKV and ToLCGV have a wider distribution. Mixed infection by two target viruses was observed in three TomLCD samples collected from Maharashtra, Punjab and Uttar Pradesh; two of these samples were infected with ToLCKV, which is prone to recombination. The results of the present study are indicative of association of uncharacterized variants or new begomoviruses with TomLCD in India, as (a) 27% of the samples found positive by the set of common primers did not amplify with species specific primers, and (b) 16% of the samples tested negative by PCR using common primers, although all the samples were collected from plants showing typical TomLCD symptoms. These plants might have been infected by some uncharacterized virus(es).