Geminiviruses and their application in biotechnology

Being a major class of single-stranded DNA viruses, geminiviruses are mostly studied due to their catastrophic infectious effect on crops. These DNA viruses are characteristic for their ability in quickly multiplying viral genetic materials without integrating into the genome of plants, which makes them ideal for developing viral vectors for plants bioengineering. Geminivirus-derived vectors can be classified into expression vectors and virus-induced gene silencing（VIGS） vectors. Details of the design, construction, application and improvements of these geminivirus vectors are summarized and discussed.

The transcriptional regulator JAZ8 interacts with the C2 protein from geminiviruses and limits the geminiviral infection in Arabidopsis

Jasmonates(JAs) are phytohormones that finely regulate critical biological processes, including plant development and defense. JASMONATE ZIM-DOMAIN(JAZ) proteins are crucial transcriptional regulators that keep JA-responsive genes in a repressed state. In the presence of JA-Ile, JAZ repressors are ubiquitinated and targeted for degradation by the ubiquitin/proteasome system,allowing the activation of downstream transcription factors and, consequently, the induction of JA-responsive genes. A growing body of evidence has shown that JA signaling is crucial in defending against plant viruses and their insect vectors. Here, we describe the interaction of C2proteins from two tomato-infecting geminiviruses from the genus Begomovirus, tomato yellow leaf curl virus(TYLCV) and tomato yellow curl Sardinia virus(TYLCSaV), with the transcriptional repressor JAZ8 from Arabidopsis thaliana and its closest orthologue in tomato, SlJAZ9. Both JAZ and C2proteins colocalize in the nucleus, forming discrete nuclear speckles. Overexpression of JAZ8did not lead to altered responses to TYLCV infection in Arabidopsis;however, knock-down of JAZ8 favors geminiviral infection. Low levels of JAZ8 likely affect the viral infection specifically,since JAZ8-silenced plants neither display obvious developmental phenotypes nor present differences in their interaction with the viral insect vector. In summary, our results show that the geminivirus-encoded C2 interacts with JAZ8 in the nucleus, and suggest that this plant protein exerts an anti-geminiviral effect.

Occurrence and distribution of geminiviruses in China

Geminiviruses are a group of plant viruses that cause severe diseases in many economically important crops worldwide, leading to devastating losses to agricultural production. Here we summarize the occurrence and distribution of geminiviruses in China,which provides valuable information for further epidemiological studies and supports the development of effective disease management strategies.

Genomic Analysis of the Natural Population of Wheat dwarf virus in Wheat from China and Hungary

During the last decade, the leafhopper transmitted Wheat dwarf virus （WDV） has become a serious problem both in northwestern China and Hungary. In order to study the molecular diversity and population structure of WDV in these two countries, 39 Chinese isolates and 16 Hungarian isolates were collected from different regions of China and Hungary, and their genomes were sequenced. All isolates belonged to the wheat strain of WDV and showed limited genetic diversity. The highest and lowest nucleotide sequence identities among isolates from China and Hungary were 99.9 and 90%, respectively. In all isolates, the lowest nucleotide sequence identity was 89.5% between MO10-1 and KP10-5, which were collected from Martonv^is^r and Kompolt, Hungary. Phylogenetic analyses showed the genome sequences of 55 WDV isolates belong to two big clades, but no clear correlation to geographical location. Population difference analyses indicated that the Chinese and Hungarian WDV populations have no significant difference. The regions in WDV genome with relatively low nucleotide diversities represented protein coding regions suggested that these regions evolved under negative selection, and might be one of the causes restricting the number of genetic variants.

Homopteran Vector Biomarkers for Efficient Circulative Plant Virus Transmission are Conserved in Multiple Aphid Species and the Whitefly Bemisia tabaci

Plant viruses in the families Luteoviridae and Geminiviridae are phloem restricted and are transmitted in a persistent,circulative manner by homopteran insects.Using fluorescence 2-D difference gel electrophoresis to compare the proteomes of F2 genotypes of Schizaphis graminum segregating for virus transmission ability,we recently discovered a panel of protein biomarkers that predict vector competency.Here we used aphid and whitefly nucleotide and expressed sequence tag database mining to test whether these biomarkers are conserved in other homopteran insects.S.graminum gene homologs that shared a high degree of predicted amino acid identity were discovered in two other aphid species and in the whitefly Bemisia tabaci.Selected reaction monitoring mass spectrometry was used to validate the expression of these biomarkers proteins in multiple aphid vector species.The conservation of these proteins in multiple insect taxa that transmit plant viruses along the circulative transmission pathway creates the opportunity to use these biomarkers to rapidly identify insect populations that are the most efficient vectors and allow them to be targeted for control prior to the spread of virus within a crop.

Two viral proteins translated from one open reading frame target different layers of plant defense

Multilayered defense responses are activated upon pathogen attack.Viruses utilize a number of strategies to maximize the coding capacity of their small genomes and produce viral proteins for infection,including suppression of host defense.Here,we reveal translation leakage as one of these strategies:two viral effec-tors encoded by tomato golden mosaic virus,chloroplast-localized C4(cC4)and membrane-associated C4(mC4),are translated from two in-frame start codons and function cooperatively to suppress defense.cC4 localizes in chloroplasts,to which it recruits NbPUB4 to induce ubiquitination of the outer membrane;as a result,this organelle is degraded,and chloroplast-mediated defenses are abrogated.However,chloroplast-localized cC4 induces the production of singlet oxygen(1O2),which in turn promotes translo-cation of the 1O2 sensor NbMBS1 from the cytosol to the nucleus,where it activates expression of the CERK1 gene.Importantly,an antiviral effect exerted by CERK1 is countered by mC4,localized at the plasma membrane.mC4,like cC4,recruits NbPUB4 and promotes the ubiquitination and subsequent degradation of CERK1,suppressing membrane-based,receptor-like kinase-dependent defenses.Importantly,this translation leakage strategy seems to be conserved in multiple viral species and is related to host range.Thisfinding suggests that stacking of different cellular antiviral responses could be an effective way to abrogate viral infection and engineer sustainable resistance to major crop viral diseases in thefield.

Geminiviral C2 proteins inhibit active autophagy to facilitate virus infection by impairing the interaction of ATG7 and ATG8

Autophagy is a conserved intracellular degradation process that plays an active role in plant response to virus infections.Here we report that geminiviruses counteract activated autophagymediated antiviral defense in plant cells through the C2 proteins they encode.We found that,in Nicotiana benthamiana plants,tomato leaf curl Yunnan virus(TLCYnV)infection upregulated the transcription levels of autophagy-related genes(ATGs).Overexpression of NbATG5,NbATG7,or NbATG8a in N.benthamiana plants decreased TLCYnV accumulation and attenuated viral symptoms.Interestingly,transgenic overexpression of NbATG7 promoted the growth of N.benthamiana plants and enhanced plant resistance to TLCYnV.We further revealed that the C2 protein encoded by TLCYnV directly interacted with the ubiquitinactivating domain of ATG7.This interaction competitively disrupted the ATG7–ATG8 binding in N.benthamiana and Solanum lycopersicum plants,thereby inhibiting autophagy activity.Furthermore,we uncovered that the C2-mediated autophagy inhibition mechanism was conserved in three other geminiviruses.In summary,we discovered a novel counter-defensive strategy employed by geminiviruses that enlists their C2 proteins as disrupters of ATG7–ATG8 interactions to defeat antiviral autophagy.

Geminivirus satellite-encoded βC1 activates UPR,induces bZIP60 nuclear export,and manipulates the expression of bZIP60 downstream genes to benefit virus infection

UPR is a conserved response in eukaryotes and can alleviate endoplasmic reticulum(ER)stresses induced by abiotic and biotic stresses.The interactions between UPR and plant RNA viruses have been documented,while the interplays between UPR and plant DNA viruses remain unknown.Using tomato yellow leaf curl China virus(TYLCCNV)and its associated betasatellite(TYLCCNB)as a model,we indicate that TYLCCNBβC1 is a major inducer of UPR and can upregulate the expression of b ZIP60,a transcription factor in Nicotiana benthamiana plants.Treatment using ER stress inducers or overexpression of Nbb ZIP60 increasesβC1 accumulation and benefits TYLCCNV/TYLCCNB infection in N.benthamiana plants,and vice versa.In the TYLCCNV/TYLCCNB-infected or theβC1-expressing cells,Nbb ZIP60 is exported from the nucleus to the nuclear periphery via the XPO1 pathway,and blocking the XPO1 pathway inhibited TYLCCNV/TYLCCNB infection.We have found that the Nbb ZIP60-regulated pro-survival genes could promote virus infection,and the pro-death gene plays a contrasting role in virus infection.This study reveals that geminivirus infection activates UPR and utilizes the up-regulated molecular chaperons to promote viral infection,and then induces the nuclear export of Nbb ZIP60 to evade plant defense response,which is a distinct virulence strategy exploited by plant pathogens.

Anthocyanin-assisted Agrobacterium infiltration for the rapid evaluation of genome editing efficiencies across multiple plant species

CRISPR-based genome editing technologies continue to drive major advances in life sciences.A major challenge for realizing widespread use of genome editing in plants and agriculture is establishing methods that enable the rapid,comprehensive,and precise evaluation of editing technologies using transient methods.Here we report a new and rapid genome editing evaluation method using Agrobacterium infiltration techniques to enable broad-spectrum,simplistic,and precise assessments of genome editing efficiencies.We employed an anthocyanin marker to facilitate visual screenings of genome-edited cells for use in adult strawberry fruits as well as tomato fruits,cotton leaves,and sugar beet leaves.Using this method,we demonstrate the ability to quickly measure genome editing efficiencies mediated by SpCas9,LbCas12a,A3A-PBE,ABE8e,and PPE.This new method will allow researchers to rapidly and easily evaluate genome editing tools across a broad spectrum of plant species,further expediting the development of genome-edited agricultural crops.

Discovery and demonstration of small circular DNA molecules derived from Chinese tomato yellow leaf curl virus

Tomato yellow leaf curl viruses belong to Begomoviruses of geminiviruses. In this work, we first found and demonstrated that the small circular DNA molecules were derived from Chinese tomato yellow leaf curl viruses (TYLCV-CHI). These small circular DNA molecules are about 1.3 kb, which are half the full-length of TYLCV-CHI DNA A. It was shown by sequence determination and analysis that there was unknown-origin sequence insertion in the middle of the small molecules. These sequences of unknown-origin were neither homologous to DNA A nor to DNA B, and were formed by recombination of virus DNA and plant DNA. Although various defective molecules contained different unknown-origin sequence insertion, all the molecules contained the intergenic region and part of the AC1 (Rep) gene. But they did not contain full ORF.

Cotton Leaf Curl Virus： Ionic Status of Leaves and Symptom Development

In the present study, the relationship between the nutritional status of leaves and the development of symptoms of cotton leaf curl virus （CLCuV） in two cotton （Gossypium hirsutum L.） cuItlvars （I.e. CIM-240 and S-12） was Investigated. The incidence of disease attack was found to be 100% In the S-12 cuItlvar and 16% in the CIM-240 cuItivar. Geminivirus particles in infected leaves were confirmed by transmission electron microscope examination of highly specific geminivirus coat protein antlsera-treated cell sap. The CLCuV Impaired the accumulation of different nutrients in both cuItivars. A marked decrease in the accumulation of Ca^2＋ and K^＋ was observed in infected leaves. However, the disease had no effect on leaf concentrations of Na^＋, N, and P. It was observed that the curling of leaf margins in CLCuV-Infected plants was associated with the leaf Ca^2＋ content; leaf curling was severe in plants with a significant reduction In Ca^2＋ content. Moreover, leaf K＆＋ content was found to be associated with resistance/susceptibility to CLCuV infection.

Tobacco RING E3 Ligase NtRFP1 Mediates Ubiquitination and Proteasomal Degradation of a Geminivirus-Encoded βC1

The βC1 protein encoded by the Tomato yellow leaf curl China virus-associated betasatellite functions as a pathogenicity determinant. To better understand the molecular basis whereby βC1 functions in pathogenicity, a yeast two-hybrid screen of a tobacco cDNA library was carried out using βC1 as the bait. The screen revealed that βC1 interacts with a tobacco RING-finger protein designated NtRFP1, which was further confirmed by the bimolecular fluorescence complementation and co-immunoprecipitation assays in Nicotiana benthamiana cells. Expression of NtRFP1 was induced by βC1, and in vitro ubiquiti- nation assays showed that NtRFP1 is a functional E3 ubiquiUn ligase that mediates βC1 ubiquitination. In addition, βC1 was shown to be ubiquitinated in vivo and degraded by the plant 26S proteasome. After viral infection, plants overexpressing NtRFP1 developed attenuated symptoms, whereas plants with silenced expression of NtRFP1 showed severe symptoms. Other lines of evidence showed that NtRFP1 attenuates βC1-induced symptoms through promoting its degradation by the 26S proteasome. Taken together, our results suggest that tobacco RING E3 ligase NtRFP1 attenuates disease symptoms by interacting with βC1 to mediate its ubiquitination and degradation via the ubiquitin/26S proteasome system.

Development and application of an efficient virus-induced gene silencing system in Nicotiana tabacum using geminivirus alphasatellite

Virus-induced gene silencing (VIGS) is a recently developed technique for characterizing the function of plant genes by gene transcript suppression and is increasingly used to generate transient loss-of-function assays. Here we report that the 2mDNA1, a geminivirus satellite vector, can induce efficient gene silencing in Nicotiana tabacum with Tobacco curly shoot virus. We have successfully silenced the β-glucuronidase (GUS) gene in GUS transgenic N. tabacum plants and the sulphur desaturase (Su) gene in five different N. tabacum cultivars. These pronounced and severe silencing phenotypes are persistent and ubiquitous. Once initiated in seedlings, the silencing phenotype lasted for the entire life span of the plants and silencing could be induced in a variety of tissues and organs including leaf, shoot, stem, root, and flower, and achieved at any growth stage. This system works well between 18-32°C. We also silenced the NtEDS1 gene and demonstrated that NtEDS1 is essential for N gene mediated resistance against Tobacco mosaic virus in N. tabacum. The above results indicate that this system has great potential as a versatile VIGS system for routine functional analysis of genes in N. tabacum.

Chinese tomato yellow leaf curl virus--a new species of geminivirus

Chinese tomato yellow leaf curl virus (TYLCV CHI) and other geminiviruses were analysed with 20 monoclonal antibodies. It was shown that TYLCV CHI is serologically close to Chinese tabacco leaf curl virus (TbLCV CHI). The fragment of TYLCV CHI DNA including the common region (CR), N terminal of coat protein gene and AV1 gene was amplified by PCR and cloned, and its DNA sequence was determined. These results showed that TYLCV CHI is different from other known geminiviruses in the world, and is a new whitefly transmitted geminivirus.

Nucieocytoplasmic Shuttling of Geminivirus C4 Protein Mediated by Phosphorylation and Myristoylation Is Critical for Viral Pathogenicity

Many geminivirus C4 proteins induce severe developmental abnormalities in plants.We previously demon- strated that Tomato leaf curl Yunnan virus (TLCYnV)C4 induces plant developmental abnormalities at least partically by decreasing the accumulation of NbSKη,an ortholog of Arabidopsis BIN2 kinase involved in the brassinosteroid signaling pathway,in the nucleus through directing it to the plasma membrane.However, the molecular mechanism by which the membrane-associated C4 modifies the localization of NbSKη in the host cell remains unclear.Here,we show that TLCYnV C4 is a nucleocytoplasmic shuttle protein,and that C4 shuttling is accompanied by nuclear export of NbSKTI.TLCYnV C4 is phosphorylated by NbSKη in the nucleus,which promotes myristoylation of the viral protein.Myristoylation of phosphorylated C4 favors its interaction with exportin-α(XPO I);which in turn facilitates nuclear export of the C4/NbSKTI complex. Supporting this model,chemical inhibition of N-myristoyltransferases or exportin-α enhanced nuclear retention of C4,and mutations of the putative phosphorylation or myristoylation sites in C4 resulted in increased nuclear retention ofrC4 and thus decreased severity of C4-induced developmental abnormalities. The impact of C4 on development is also lessened when a nuclear localization signal or a nuclear export signal is added to its C-terminus,restricting it to a specific cellular niche and therefore impairing nucleocytoplasmic shuttling.Taken together,our results suggest that nucleocytoplasmic shuttling of TLCYnV C4,enabled by phosphorylation by NbSKη,myristoylation,and interaction with exportin-α is critical for its function as a pathogenicity factor.

Chinese squash leaf curl virus: a new whitefly-transmitted geminivirus

Coat protein (CP) gene of the Chinese squash !eaf curl virus (SqLCV-C) was amplified through PC’R, cloned and completely sequenced. Based on the comparisons at the levels of both CP gene nucleotide and CP -deduced amino acid sequences with other geminiviruses, SqLCV-C is confirmed to be distinct from the American squash leaf curl virus (SqLCV-E). It is a new geminivirus transmitted by whitefly Bemisia tabaci, which infects dicotyledonous plants and is more closely related to the Indian cassava mosaic virus (ICMV).

Functional analysis of a novel βV1 gene identified in a geminivirus betasatellite

Betasatellites(DNAβ)are circular ssDNA molecules that are associated with monopartite geminiviruses and exert a positive effect on the viral infection.Betasatellites encode one protein,namedβC1,on the complementary strand;βC1 functions as a pathogenicity factor and RNA silencing suppressor.In this report,we describe the identification of another betasatellite-encoded protein,βV1,which also contributes to symptom development.TheβV1 open reading frame can be found on the viral strand of approximately 40%of reported betasatellite sequences,and is conserved in position and sequence.The presence of theβV1 transcript was observed in plants infected with Tomato yellow leaf curl China virus(TYLCCNV)along with its associated betasatellite Tomato yellow leaf curl China betasatellite(TYLCCNB).Mutant viruses unable to produceβV1 showed reduced virulence and decreased viral load.Ectopic expression of the TYLCCNB-βV1 gene in Nicotiana benthamiana plants from a PVX-based vector resulted in leaf mosaic and chlorosis.We further demonstrated that theβV1 protein could elicit hypersensitive response(HR)-type cell death in N.benthamiana leaves.Our results uncover a novel betasatellite-encoded protein that contributes to the virus infection,and this discover gives us a more complete view of the plant-geminivirus interaction landscape.

Preliminary Study on Molecular Evolution of Tobacco Leaf Curl Geminivirus

Leaf curl is a serious disease of tobacco in tropical and subropical regions. Itscausative agent is tobacco leaf curl geminivirus (TLCV)which can be transmitted bywhitefly (Bemisia tabaci) and infect many other plant species of Solanaceae andCaricaceae.The occurrence, host range and insect vector of TbLCV as well as itsserological reaction with monoclonal antibody of African cassava mosaic virus havebeen reported in China, and more recently, tobacco leaf curl disease has becomeendemic in southern areas. Here we first present the nucleotide sequence ofTbLCV coat protein (CP) gene and molecular evolutionary relationship with

The Ever-Expanding Role of C4/AC4 in Geminivirus Infection: Punching above Its Weight?

Geminiviruses (family Geminiviridae) are among the most devastating plant pathogens worldwide, causing heavy losses on food and cash crops and representing a threat to global food security and sustainability. These viruses have very compact genomes comprising one (monopartite) or two (bipartite) circular singlestranded DNA (ssDNA) molecules, each ranging from 2.6 to 2.8 kb.

Expressing activity of promoter elements of large intergenic region from cotton leaf curl virus in host plant

Cotton leaf curl virus (CLCuV) is a type of single-stranded DMA virus, belonging to geminivirus of subgroup Ⅲ. In order to determine the function of CLCuV large intergenic region (LIR), total DNA of CLCuV-infected cotton leaves was used as template, and fragment of LIR was obtained by PCR and inserted into clone vector. The fragment of LIR was fused with gus reporter gene and nos terminator in the orientation of transcription of virion sense and complementary sense respectively, and the plant expression vectors were constructed. GUS activity of Agrobacte-rium-mediated transgenic tobacco was measured. The result indicated that LIR showed strong promoter activity in complementary sense gene orientation. Average GUS activity of the complementary sense promoter was 5-6 times that of CaMV 35S promoter, and the highest GUS activity of individual plant was ten times of that of CaMV 35S promoter. Histochemical localization confirmed its activity in both mesophyll and vascular tissues. Activity of virion sense of LIR was rather low. Thus LIR isolated from CLCuV could be used as a novel strong promoter in plant genetic manipulation.