Tomato Yellow Leaf Curl Virus Acquisition Research Articles

Evaluating Acylsugars-Mediated Resistance in Tomato against Bemisia tabaci and Transmission of Tomato Yellow Leaf Curl Virus.

Simple SummaryThe sweetpotato whitefly, Bemisia tabaci, besides causing feeding-related injuries, also transmits the economically devastating tomato yellow leaf curl virus (TYLCV) to tomato plants. The main management options include host resistance and insecticides. Host resistance is imparted against TYLCV and not against the vector. Incorporating host resistance against whiteflies could broaden management options. Acylsugars are secondary metabolites exuded from trichomes of wild solanums that can negatively impact numerous herbivores, including whiteflies. This study examined the effects of acylsugar-producing tomato lines with fatty acid quantitative trait loci introgressions from Solanum pennellii LA716 into cultivated tomato on whitefly preference and fitness, and whitefly-mediated TYLCV inoculation and subsequent TYLCV acquisition. Overall, the acylsugar-producing lines negatively affected whitefly preference and fitness in comparison with the non-acylsugar hybrid. Acylsugars’ mediated antixenosis and antibiosis effects against whiteflies were documented. Acylsugar-producing lines also reduced whitefly-mediated TYLCV inoculation and subsequent TYLCV acquisition by whiteflies in comparison with the non-acylsugar hybrid. These results suggest that acylsugar-mediated resistance against whiteflies could complement already existing TYLCV resistance in tomato cultivars/hybrids and could reduce the heavy reliance on chemical control of whiteflies.The sweetpotato whitefly, Bemisia tabaci, is a major pest of cultivated tomato. Whitefly feeding-related injuries and transmission of viruses including tomato yellow leaf curl virus (TYLCV) cause serious losses. Management strategy includes planting resistant cultivars/hybrids. However, TYLCV resistance is incomplete and whiteflies on TYLCV-resistant cultivars/hybrids are managed by insecticides. Acylsugars’-mediated resistance against whiteflies has been introgressed from wild solanums into cultivated tomato. This study evaluated acylsugar-producing tomato lines with quantitative trait loci (QTL) containing introgressions from Solanum pennellii LA716, known to alter acylsugars’ levels or chemistry. Evaluated acylsugar-producing lines were the benchmark line CU071026, QTL6/CU071026—a CU071026 sister line with QTL6, and three other CU071026 sister lines with varying QTLs—FA2/CU71026, FA7/CU071026, and FA2/FA7/CU071026. Non-acylsugar tomato hybrid Florida 47 (FL47) was also evaluated. Acylsugars’ amounts in FA7/CU071026 and FA2/FA7/CU071026 were 1.4 to 2.2 times greater than in other acylsugar-producing lines. Short chain fatty acid, i-C5, was dominant in all acylsugar-producing lines. Long chain fatty acids, n-C10 and n-C12, were more abundant in FA7/CU071026 and FA2/FA7/CU071026 than in other acylsugar-producing lines. Whiteflies preferentially settled on non-acylsugar hybrid FL47 leaves over three out of five acylsugar-producing lines, and whiteflies settled 5 to 85 times more on abaxial than adaxial leaf surface of FL47 than on acylsugar-producing lines. Whiteflies’ survival was 1.5 to 1.9 times lower on acylsugar-producing lines than in FL47. Nevertheless, whiteflies’ developmental time was up to 12.5% shorter on acylsugar-producing lines than on FL47. TYLCV infection following whitefly-mediated transmission to acylsugar-producing lines was 1.4 to 2.8 times lower than FL47, and TYLCV acquisition by whiteflies from acylsugar-producing lines was up to 77% lower than from FL47. However, TYLCV accumulation in acylsugar-producing lines following infection and TYLCV loads in whiteflies upon acquisition from acylsugar-producing lines were not different from FL47. Combining TYLCV resistance with acylsugars’-mediated whitefly resistance in cultivated tomato could substantially benefit whiteflies and TYLCV management.

Combined infection with Tomato yellow leaf curl virus and Rickettsia influences fecundity, attraction to infected plants and expression of immunity-related genes in the whitefly Bemisia tabaci.

We have recently shown that Rickettsia, a secondary facultative bacterial symbiont that infects the whitefly B. tabaci is implicated in the transmission of Tomato yellow leaf curl virus (TYLCV). Infection with Rickettsia improved the acquisition and transmission of the virus by B. tabaci adults. Here we performed a transcriptomic analysis with Rickettsia-infected and uninfected B. tabaci adults before and after TYLCV acquisition. The results show a dramatic and specific activation of the immune system in the presence of Rickettsia before TYLCV acquisition. However, when TYLCV was acquired, it induced massive activation of gene expression in the Rickettsia uninfected population, whereas in the Rickettsia-infected population the virus induced massive down-regulation of gene expression. Fitness and choice experiments revealed that while Rickettsia-infected whiteflies are always more attracted to TYLCV-infected plants, this attraction is not always beneficiary for their offspring. These studies further confirm the role of Rickettsia in many aspects of B. tabaci interactions with TYLCV, and possibly serves as an important factor in the dissemination of the virus.

Persistently Transmitted Viruses Restrict the Transmission of Other Viruses by Affecting Their Vectors.

Diverse pathogens, plant hosts, insect vectors, and non-vector herbivores coexist and interact in natural systems. An example is the cooccurrence of insects Bemisia tabaci Q and Frankliniella occidentalis and the pathogens tomato yellow leaf curl virus (TYLCV) and tomato spotted wilt virus (TSWV) on the same plant. In addition, both TYLCV and TSWV are persistently transmitted in these insect species. However, TSWV reduces the fitness of B. tabaci Q; therefore, we investigated whether TSWV affects the transmission of TYLCV to tomato. Both TYLCV and TSWV are persistently transmitted. Although B. tabaci Q cannot transmit TSWV, we found that this insect species is able to acquire and retain this virus serotype, indicating that the effects of TSWV on TYLCV transmission in the current study result from effects on the vector. The acquisition, retention, and transmission of TYLCV by B. tabaci Q were reduced when the insect vector contained TSWV. Additionally, the TYLCV acquisition and transmission by B. tabaci Q were reduced when the host plant was inoculated with TSWV before TYLCV or simultaneously with TYLCV. We also found that F. occidentalis fecundity and transmission of TSWV were reduced when F. occidentalis contained TYLCV. Our findings are consistent with the hypothesis that persistently transmitted viruses can restrict the transmission of other viruses by affecting their insect vectors.

Implication of the Whitefly Bemisia tabaci Cyclophilin B Protein in the Transmission of Tomato yellow leaf curl virus.

Tomato yellow leaf curl virus (TYLCV) is a single-stranded (ssDNA) begomoviruses that causes severe damage to tomato and several other crops worldwide. TYLCV is exclusively transmitted by the sweetpotato whitefly, Bemisia tabaci in a persistent circulative and propagative manner. Previous studies have shown that the transmission, retention, and circulation of TYLCV in its vector involves interaction with insect and endosymbiont proteins, which aid in the transmission of the virus, or have a protective role in response to the presence of the virus in the insect body. However, only a low number of such proteins have been identified. Here, the role of B. tabaci Cyclophilin B (CypB) in the transmission of TYLCV protein was investigated. Cyclophilins are a large family of cellular prolyl isomerases that have many molecular roles including facilitating protein-protein interactions in the cell. One cyclophilin protein has been implicated in aphid-luteovirus interactions. We demonstrate that the expression of CypB from B. tabaci is altered upon TYLCV acquisition and retention. Further experiments used immunocapture-PCR and co-immunolocalization and demonstrated a specific interaction and colocalization between CypB and TYLCV in the the midgut, eggs, and salivary glands. Membrane feeding of anti-CypB antibodies and TYLCV-infected plants showed a decrease in TYLCV transmission, suggesting a critical role that CypB plays in TYLCV transmission. Further experiments, which used membrane feeding with the CypB inhibitor Cyclosporin A showed decrease in CypB-TYLCV colocalization in the midgut and virus transmission. Altogether, our results indicate that CypB plays an important role in TYLCV transmission by B. tabaci.

Temporal Effects of a Begomovirus Infection and Host Plant Resistance on the Preference and Development of an Insect Vector, Bemisia tabaci, and Implications for Epidemics.

Persistent plant viruses, by altering phenotypic and physiological traits of their hosts, could modulate the host preference and fitness of hemipteran vectors. A majority of such modulations increase vector preference for virus-infected plants and improve vector fitness, ultimately favouring virus spread. Nevertheless, it remains unclear how these virus-induced modulations on vectors vary temporally, and whether host resistance to the pathogen influences such effects. This study addressed the two questions using a Begomovirus-whitefly-tomato model pathosystem. Tomato yellow leaf curl virus (TYLCV) -susceptible and TYLCV-resistant tomato genotypes were evaluated by whitefly-mediated transmission assays. Quantitative PCR revealed that virus accumulation decreased after an initial spike in all genotypes. TYLCV accumulation was less in resistant than in susceptible genotypes at 3, 6, and 12 weeks post inoculation (WPI). TYLCV acquisition by whiteflies over time from resistant and susceptible genotypes was also consistent with virus accumulation in the host plant. Furthermore, preference assays indicated that non-viruliferous whiteflies preferred virus-infected plants, whereas viruliferous whiteflies preferred non-infected plants. However, this effect was prominent only with the susceptible genotype at 6 WPI. The development of whiteflies on non-infected susceptible and resistant genotypes was not significantly different. However, developmental time was reduced when a susceptible genotype was infected with TYLCV. Together, these results suggest that vector preference and development could be affected by the timing of infection and by host resistance. These effects could play a crucial role in TYLCV epidemics.

Transmission of Tomato Yellow Leaf Curl Virus by Bemisia tabaci as Affected by Whitefly Sex and Biotype.

Bemisia tabaci is a serious pest of vegetables and other crops worldwide. The most damaging and predominant B. tabaci biotypes are B and Q, and both are vectors of tomato yellow leaf curl virus (TYLCV). Previous research has shown that Q outperforms B in many respects but comparative research is lacking on the ability of B and Q to transmit viruses. In the present study, we tested the hypothesis that B and Q differ in their ability to transmit TYLCV and that this difference helps explain TYLCV outbreaks. We compared the acquisition, retention, and transmission of TYLCV by B and Q females and males. We found that Q females are more efficient than Q males, B females, and B males at TYLCV acquisition and transmission. Although TYLCV acquisition and transmission tended to be greater for B females than B males, the differences were not statistically significant. Based on electrical penetration graphs determination of phloem sap ingestion parameters, females fed better than males, and Q females fed better than Q males, B females, or B males. These results are consistent with the occurrences of TYLCV outbreaks in China, which have been associated with the spread of Q rather than B.

Expression and functional characterisation of a soluble form of Tomato yellow leaf curl virus coat protein

Tomato yellow leaf curl virus (TYLCV), a member of the genus Begomovirus within the family Geminiviridae, is an important pathogen of tomato in many tropical, subtropical and temperate regions. TYLCV is exclusively transmitted by the whitefly Bemisia tabaci in a circulative manner. The viral coat protein (CP) has been assumed to play important roles in the entry of TYLCV into the insect midgut cells. Testing the hypothesis that CP plays an important role in TYLCV acquisition by B. tabaci, a soluble form of the CP was expressed and purified. The purified recombinant CP made it possible to examine the function of TYLCV CP without other viral proteins. In an in vivo binding assay, specific binding of TYLCV CP to B. tabaci midguts was detected when purified CP was fed to B. tabaci. In addition, real-time polymerase chain reaction analysis of virus titre revealed that B. tabaci fed with purified CP had reduced the level of virus in their midgut compared with those fed with bovine serum albumin or maltose-binding protein. These results suggest that binding of TYLCV CP to the B. tabaci midgut specifically inhibits virus acquisition. The findings that TYLCV CP binds to B. tabaci midguts and decreases virus acquisition provide direct evidence that CP mediates the attachment of TYLCV to receptors on the epithelial cells of the B. tabaci midgut.

Implication of Bemisia tabaci heat shock protein 70 in Begomovirus-whitefly interactions.

The whitefly Bemisia tabaci (Gennadius) is a major cosmopolitan pest capable of feeding on hundreds of plant species and transmits several major plant viruses. The most important and widespread viruses vectored by B. tabaci are in the genus Begomovirus, an unusual group of plant viruses owing to their small, single-stranded DNA genome and geminate particle morphology. B. tabaci transmits begomoviruses in a persistent circulative nonpropagative manner. Evidence suggests that the whitefly vector encounters deleterious effects following Tomato yellow leaf curl virus (TYLCV) ingestion and retention. However, little is known about the molecular and cellular basis underlying these coevolved begomovirus-whitefly interactions. To elucidate these interactions, we undertook a study using B. tabaci microarrays to specifically describe the responses of the transcriptomes of whole insects and dissected midguts following TYLCV acquisition and retention. Microarray, real-time PCR, and Western blot analyses indicated that B. tabaci heat shock protein 70 (HSP70) specifically responded to the presence of the monopartite TYLCV and the bipartite Squash leaf curl virus. Immunocapture PCR, protein coimmunoprecipitation, and virus overlay protein binding assays showed in vitro interaction between TYLCV and HSP70. Fluorescence in situ hybridization and immunolocalization showed colocalization of TYLCV and the bipartite Watermelon chlorotic stunt virus virions and HSP70 within midgut epithelial cells. Finally, membrane feeding of whiteflies with anti-HSP70 antibodies and TYLCV virions showed an increase in TYLCV transmission, suggesting an inhibitory role for HSP70 in virus transmission, a role that might be related to protection against begomoviruses while translocating in the whitefly.

Whitefly Population Dynamics and Evaluation of Whitefly-Transmitted Tomato Yellow Leaf Curl Virus (TYLCV)-Resistant Tomato Genotypes as Whitefly and TYLCV Reservoirs

Sweetpotato whitefly, Bemisia tabaci (Gennadius), and whitefly-transmitted tomato yellow leaf curl virus (TYLCV) are major threats to tomato production in the southeastern United States. TYLCV was introduced to Florida from the Caribbean islands and has spread to other southern states of the United States. In Georgia, in recent years, the incidence of TYLCV has been steadily increasing. Studies were conducted to monitor population dynamics of whiteflies in the vegetable production belt of Georgia, to evaluate TYLCV-resistant genotypes against whiteflies and TYLCV, and to assess the potential role of resistant genotypes in TYLCV epidemiology. Monitoring studies indicated that the peak incidence of whiteflies varied seasonally from year to year. In general, whitefly populations were not uniformly distributed. Tomato genotypes exhibited minor differences in their ability to support whitefly populations. TYLCV symptoms were visually undetectable in all but one resistant genotype. The infection rates (visually) in susceptible genotypes ranged from 40 to 87%. Greenhouse inoculations with viruliferous whiteflies followed by polymerase chain reaction (PCR) indicated that up to 100% of plants of resistant genotypes were infected, although predominantly symptomless. TYLCV acquisition by whiteflies from TYLCV-infected genotypes was tested by PCR; TYLCV acquisition rates from resistant genotypes were less than from susceptible genotypes. Nevertheless, this difference did not influence TYLCV transmission rates from resistant to susceptible genotypes. Results emphasize that resistant genotypes can serve as TYLCV and whitefly reservoirs and potentially influence TYLCV epidemics.

Upregulation of temperature susceptibility in Bemisia tabaci upon acquisition of Tomato yellow leaf curl virus (TYLCV)

Acquisition of plant viruses has various effects on physiological mechanisms in vector insects. Bemisia tabaci is the only known vector of Tomato yellow leaf curl virus (TYLCV), which is a serious virus affecting tomato cultivars. In this study, the lifespan of Q1 biotype was compared between non-viruliferous (NV) and TYLCV-viruliferous (V) whiteflies. Total lifespan from egg to adult death of NV whiteflies was 62.54days but 10.64days shorter in V whiteflies. We investigated the temperature susceptibility of B. tabaci by comparing mortalities as well as heat shock protein (hsp) mRNA levels between NV and V whiteflies. For this, NV and V whiteflies were exposed for either 1 or 3h at 4, 25, and 35°C. The mortality of V whiteflies was higher than NV ones following exposure at either 4 or 35°C, but there was no significant difference at 25°C. Analysis of the expression level of heat shock protein (hsp) genes using quantitative real-time PCR showed that both cold and heat shock treatments stimulated higher expression of hsps (hsp40, hsp70, and hsp90) at various rates in V whiteflies than NV ones, but there was no difference at 25°C. All together, our results show that TYLCV acquisition accelerated the developmental rate and increased susceptibility to thermal stress in B. tabaci. Therefore, this modification may result in reduced vector longevity due to increased metabolic energy utilization. Our results provide insights into the complex interaction between vector fitness and thermal stress in relation to the acquisition and transmission of plant viruses.

Pcr-amplification of tomato yellow leaf curl virus (TYLCV) DNA from squashes of plants and whitefly vectors: Application to the study of TYLCV acquisition and transmission

DNA of tomato yellow leaf curl virus (TYLCV), a geminivirus transmitted by the whitefly Bemisia tabaci, was amplified from squashes of infected tomato plants and of viruliferous vectors using the polymerase chain reaction (PCR). Samples of infected tissues as small as 1 mm2 were squashed onto a nylon membrane. A 1 × 2 mm strip containing the squash was introduced into a 25 µl PCR reaction mix. The reaction products were subjected to gel electrophoresis, blotted and hybridized with a radiolabeled virus-specific DNA probe. TYLCV DNA was amplified from squashes of leaves, roots, and stem of infected tomato and from individual viruliferous whiteflies. The same squash could be used several times to amplify different virus DNA fragments with various sets of primers. Thus plant and insect squashes can be used as templates for the amplification of geminiviral DNA with no need to prepare tissue extracts or purify nucleic acids. The squash-PCR procedure was applied to study whitefly transmission of TYLCV. Tomato plants were inoculated by placing a single viruliferous insect in the center of a young leaflet. In some plants TYLCV DNA was detected at the site of inoculation as early as 5 min after the beginning of the access feeding and in all plants after 30 min. The squash-PCR procedure also was applied to the study of TYLCV acquisition by the insect vector. TYLCV DNA was detected in the head of whiteflies as early as 5 min after the beginning of the access feeding on infected tomato plants. Viral DNA was detected in the thorax after 10 min and in the abdomen after 25 min.