Begomoviral ßC1 orchestrates organellar genomic instability to augment viral infection.

Chloroplast is the site for transforming light energy to chemical energy. It also acts as a production unit for a variety of defense-related molecules. These defense moieties are necessary to mount a successful counter defense against pathogens, including viruses. Previous studies indicated disruption of chloroplast homeostasis as a basic strategy of Begomovirus for its successful infection leading to the production of vein-clearing, mosaic, and chlorotic symptoms in infected plants. Although begomoviral pathogenicity determinant protein Beta C1 (ßC1) was implicated for pathogenicity, the underlying mechanism was unclear. Here we show that, begomoviral ßC1 directly interferes with the host plastid homeostasis. ßC1 induced DPD1, an organelle-specific nuclease, implicated in nutrient salvage and senescence, as well as modulated the function of a major plastid genome maintainer protein RecA1, to subvert plastid genome. We show that ßC1 was able to physically interact with bacterial RecA and its plant homolog RecA1, resulting in its altered activity. We observed that knocking-down DPD1 during virus infection significantly reduced virus-induced necrosis. These results indicate the presence of a strategy in which a viral protein alters host defense by targeting modulators of chloroplast DNA. We predict that the mechanism identified here might have similarities in other plant-pathogen interactions.

Betasatellite-encoded ßC1 protein regulates helper virus accumulation by interfering with the ATP hydrolysis activity of geminivirus-encoded replication initiator protein.

Geminivirus-betasatellite disease complexes are an epidemic threat to the majority of economically important crops across the world. Plant virus satellites including betasatellites are maintained by their associated helper virus. Geminivirus-betasatellites influence viral pathogenesis by substantially increasing or decreasing their helper virus accumulation. In the present study, we attempted to understand the mechanistic details of the geminivirus-betasatellite interaction. Here, we used tomato leaf curl Gujarat virus (ToLCGV) and tomato leaf curl Patna betasatellite (ToLCPaB) as a model system. This study reveals that ToLCGV can efficiently trans-replicate ToLCPaB in Nicotiana benthamiana plants, but ToLCPaB greatly reduced the accumulation of its helper virus DNA. For the first time, we have identified that the ToLCPaB-encoded ßC1 protein is able to interact with ToLCGV-encoded replication initiator protein (Rep). In addition, we demonstrate that the C-terminal region of ßC1 interacts with the C-terminus of Rep (RepC) protein. Our previous study had established that ßC1 proteins encoded by diverse betasatellites possess a novel ATP hydrolysis activity and the conserved lysine/arginine residues at positions 49 and 91 are necessary for this function. Here, we show that mutating lysine at positions 49 to alanine of ßC1 (ßC1K49A) protein did not affect its ability to interact with RepC protein. Biochemical studies performed with ATP hydrolysis activity-deficient K49A mutated ßC1 (ßC1K49A) and RepC proteins revealed that Rep-ßC1 interaction interferes with the ATP hydrolysis activity of Rep protein. Further, we demonstrate that ßC1 protein is able to interact with D227A and D289A mutated RepC proteins but not with D262A, K272A or D286A mutated RepC proteins, suggesting that the ßC1-interacting region of Rep protein encompasses its Walker-B and B' motifs. The results of docking studies supported that the ßC1-interacting region of Rep protein encompasses its motifs associated with ATP binding and ATP hydrolysis activities. Docking studies also provided evidence that the Rep-ßC1 interaction interferes with the ATP binding activity of Rep protein. Together, our findings suggest that ßC1 protein regulates helper virus accumulation by interfering with the ATP hydrolysis activity of helper virus Rep protein.

Geminiviral betasatellites: critical viral ammunition to conquer plant immunity.

Geminiviruses have mastered plant cell modulation and immune invasion to ensue prolific infection. Encoding a relatively small number of multifunctional proteins, geminiviruses rely on satellites to efficiently re-wire plant immunity, thereby fostering virulence. Among the known satellites, betasatellites have been the most extensively investigated. They contribute significantly to virulence, enhance virus accumulation, and induce disease symptoms. To date, only two betasatellite proteins, ßC1, and ßV1, have been shown to play a crucial role in virus infection. In this review, we offer an overview of plant responses to betasatellites and counter-defense strategies deployed by betasatellites to overcome those responses.

Geminivirus Betasatellite-Encoded ßC1 Protein Exhibits Novel ATP Hydrolysis Activity That Influences Its DNA-Binding Activity and Viral Pathogenesis.

Plant virus satellites are maintained by their associated helper viruses, and satellites influence viral pathogenesis. Diseases caused by geminivirus-betasatellite complexes can become epidemics and therefore have become a threat to economically important crops across the world. Here, we identified a novel molecular function of the betasatellite-encoded pathogenicity determinant ßC1. The tomato leaf curl Patna betasatellite (ToLCPaB)-encoded ßC1 protein was found to exhibit novel ATPase activity in the presence of the divalent metal ion cofactor MgCl2. Moreover, ATPase activity was confirmed to be ubiquitously displayed by ßC1 proteins encoded by diverse betasatellites. Mutational and sequence analysis showed that conserved lysine/arginine residues at positions 49/50 and 91 of ßC1 proteins are essential for their ATPase activity. Biochemical studies revealed that the DNA-binding activity of the ßC1 protein was interfered with by the binding of ATP to the protein. Mutating arginine 91 of ßC1 to alanine reduced its DNA-binding activity. The results of docking studies provided evidence for an overlap of the ATP-binding and DNA-binding regions of ßC1 and for the importance of arginine 91 for both ATP-binding and DNA-binding activities. A mutant betasatellite with a specifically ßC1-ATPase dominant negative mutation was found to induce symptoms on Nicotiana benthamiana plants similar to those induced by wild-type betasatellite infection. The ATPase function of ßC1 was found to be negatively associated with geminivirus-betasatellite DNA accumulation, despite the positive influence of this ATPase function on the accumulation of replication-associated protein (Rep) and ßC1 transcripts. IMPORTANCE Most satellites influence the pathogenesis of their helper viruses. Here, we characterized the novel molecular function of ßC1, a nonstructural pathogenicity determinant protein encoded by a betasatellite. We demonstrated the display of ATPase activity by this ßC1 protein. Additionally, we confirmed the ubiquitous display of ATPase activity by ßC1 proteins encoded by diverse betasatellites. The lysine/arginine residues conserved at positions 49 and 91 of ßC1 were found to be crucial for its ATPase function. DNA-binding activity of ßC1 was found to be reduced in the presence of ATP. Inhibition of ATPase activity of ßC1 in the presence of an excess concentration of cold ATP, GTP, CTP, or UTP suggested that the purified ßC1 can also hydrolyze other cellular nucleoside triphosphates (NTPs) besides ATP in vitro. These results established the importance of the ATPase and DNA-binding activities of the ßC1 protein in regulating geminivirus-betasatellite DNA accumulation in the infected plant cell.

Stability of Begomoviral pathogenicity determinant ßC1 is modulated by mutually antagonistic SUMOylation and SIM interactions.

BACKGROUND: To successfully invade new hosts, plant viruses must break host resistance and be competent to move within and between plant cells. As a means, viral proteins known as pathogenicity determinants have evolved to coordinate a network of protein interactions. The ßC1 protein encoded by specific geminiviral satellites acts as a key pathogenicity determinant for this disease-causing family of plant viruses. Post-translational modifications (PTMs) such as ubiquitination and phosphorylation of the ßC1 protein have been shown to occur in diverse viruses. However, the relevance of these and other layers of PTMs in host-geminiviral interactions has not been fully understood. RESULTS: Here we identified the significance of a novel layer of PTMs in the ßC1 protein of Synedrella yellow vein clearing virus (SyYVCV), a newly identified member of the Begomovirus genus of Geminiviruses. This protein has conserved SUMOylation and SUMO-interacting motifs (SIMs), and we observed SUMOylation of SyYVCV ßC1 in host plants as a defensive strategy against ubiquitin-mediated degradation. Counteracting this, SIMs encoded in ßC1 mediate the degradation of ßC1; however, both these PTMs are essential for the function of ßC1 protein since SIM and SUMOylation motif mutants failed to promote pathogenicity and viral replication in vivo. SUMOylation in different motifs of ßC1 led to functionally distinct outcomes, regulating the stability and function of the ßC1 protein, as well as increased global SUMOylation of host proteins. CONCLUSION: Our results indicate the presence of a novel mechanism mediating a fine balance between defence and counter-defence in which a SIM site is competitively sought for degradation and, as a counter-defence, ßC1 undergoes SUMOylation to escape from its degradation.

Journey of begomovirus betasatellite molecules: from satellites to indispensable partners.

Betasatellites are a group of circular, single-stranded DNA molecules that are frequently found to be associated with monopartite begomoviruses of the family Geminiviridae. Betasatellites require their helper viruses for replication, movement, and encapsidation and they are often essential for induction of typical disease symptoms. The ßC1 protein encoded by betasatellites is multifunctional that participates in diverse cellular events. It interferes with several cellular processes like normal development, chloroplasts, and innate immune system of plants. Recent research has indicated ßC1 protein interaction with cellular proteins and its involvement in modulation of the host's cell cycle and symptom determination. This article focuses on the functional mechanisms of ßC1 and its interactions with other viral and host proteins.

Heat shock transcriptional factor mediates mitochondrial unfolded protein response.

For maintenance of cytoplasmic protein quality control (PQC), cytoplasmic heat shock proteins (HSPs) negatively control heat shock transcriptional factor (HSF) in a negative feedback loop. However, how mitochondrial protein quality control (mtPQC) is maintained is largely unknown. Here we present evidence that HSF directly monitors mtPQC in the budding yeast Saccharomyces cerevisiae. Mitochondrial HSP70 (Ssc1) negatively regulated HSF activity. Importantly, HSF was localized not only in the nucleus but also on mitochondria. The mitochondrial localization of HSF was increased by heat shock and compromised by SSC1 overexpression. Furthermore, the mitochondrial protein translocation system downregulated HSF activity. Finally, mtPQC modulated the mtHSP genes SSC1 and MDJ1 via HSF, and SSC1 overexpression compromised mitochondrial function. These findings illustrate a model in which HSF directly monitors mtPQC.

A geminivirus betasatellite damages the structural and functional integrity of chloroplasts leading to symptom formation and inhibition of photosynthesis.

Geminivirus infection often causes severe vein clearing symptoms in hosts. Recently a betasatellite has emerged as a key regulator of symptom induction. To understand the host-betasatellite interactions in the process of symptom development, a systematic study was carried out involving symptoms induced by a betasatellite associated with radish leaf curl disease (RaLCB) in Nicotiana benthamiana. It has been found that ßC1 protein localized to chloroplasts of host cells, and RaLCB lacking ßC1, which failed to produce symptoms, had no effect on chloroplast ultrastructure. Vein flecking induced by transiently expressed ßC1 was associated with chloroplast ultrastructure. In addition, the betasatellite down-regulates expression of genes involved in chlorophyll biosynthesis as well as genes involved in chloroplast development and plastid translocation. Interestingly, the expression of key host genes involved in chlorophyll degradation remains unaffected. Betasatellite infection drastically reduced the numbers of active reaction centres and the plastoquinol pool size in leaves exhibiting vein clearing symptoms. Betasatellite-mediated impediments at different stages of chloroplast functionality affect the photosynthetic efficiency of N. benthamiana. To the best of the authors' knowledge, this is the first evidence of a chloroplast-targeting protein encoded by a DNA virus which induces vein clearing and structurally and functionally damages chloroplasts in plants.

Geminivirus satellite-encoded ßC1 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 TYLCCNB ßC1 is a major inducer of UPR and can upregulate the expression of bZIP60, a transcription factor in Nicotiana benthamiana plants. Treatment using ER stress inducers or overexpression of NbbZIP60 increases ßC1 accumulation and benefits TYLCCNV/TYLCCNB infection in N. benthamiana plants, and vice versa. In the TYLCCNV/TYLCCNB-infected or the ßC1-expressing cells, NbbZIP60 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 NbbZIP60-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 NbbZIP60 to evade plant defense response, which is a distinct virulence strategy exploited by plant pathogens.

Temporal Dynamic of the Ratio between Monopartite Begomoviruses and Their Associated Betasatellites in Plants, and Its Modulation by the Viral Gene ßC1.

The begomovirus-betasatellite complex constantly threatens crops in Asia. However, the quantitative relationship between begomoviruses and betasatellites remains largely unknown. The quantities of tobacco curly shoot virus (TbCSV) and its betasatellite (TbCSB) and their ratio varied significantly in initial infection, and thereafter, the ratio tended to become constant. The TbCSB/TbCSV ratio in agrobacteria inoculum significantly affected that in plants in the initial infection but not thereafter. Null-mutation of ßC1 that encodes a multifunctional protein important for pathogenesis in TbCSB significantly reduced the TbCSB/TbCSV ratio in plants. Viral inoculum plants with higher TbCSB/TbCSV ratios promoted whitefly transmission of the virus. The expression of AV1 encoded by TbCSV, ßC1 encoded by TbCSB and the ßC1/AV1 ratio varied significantly in the initial infection and thereafter the ratio tended to become constant. Additionally, the temporal dynamics of the ratio between another begomovirus and its betasatellite was similar to that of TbCSV and was positively regulated by ßC1. These results indicate that the ratio between monopartite begomoviruses and betasatellites tend to become constant as infection progresses, and is modulated by ßC1, but a higher betasatellite/begomovirus ratio in virally inoculated plants promotes virus transmission by whiteflies. Our findings provide novel insights into the association between begomoviruses and betasatellites.

Diverse Begomoviruses Evolutionarily Hijack Plant Terpenoid-Based Defense to Promote Whitefly Performance.

Arthropod-borne pathogens and parasites are major threats to human health and global agriculture. They may directly or indirectly manipulate behaviors of arthropod vector for rapid transmission between hosts. The largest genus of plant viruses, Begomovirus, is transmitted exclusively by whitefly (Bemisia tabaci), a complex of at least 34 morphologically indistinguishable species. We have previously shown that plants infected with the tomato yellowleaf curl China virus (TYLCCNV) and its associated betasatellite (TYLCCNB) attract their whitefly vectors by subverting plant MYC2-regulated terpenoid biosynthesis, therefore forming an indirect mutualism between virus and vector via plant. However, the evolutionary mechanism of interactions between begomoviruses and their whitefly vectors is still poorly understood. Here we present evidence to suggest that indirect mutualism may happen over a millennium ago and at present extensively prevails. Detailed bioinformatics and functional analysis identified the serine-33 as an evolutionary conserved phosphorylation site in 105 of 119 Betasatellite species-encoded ßC1 proteins, which are responsible for suppressing plant terpenoid-based defense by interfering with MYC2 dimerization and are essential to promote whitefly performance. The substitution of serine-33 of ßC1 proteins with either aspartate (phosphorylation mimic mutants) or cysteine, the amino acid in the non-functional sßC1 encoded by Siegesbeckia yellow vein betasatellite SiYVB) impaired the ability of ßC1 functions on suppression of MYC2 dimerization, whitefly attraction and fitness. Moreover the gain of function mutation of cysteine-31 to serine in sßC1 protein of SiYVB restored these functions of ßC1 protein. Thus, the dynamic phosphorylation of serine-33 in ßC1 proteins helps the virus to evade host defense against insect vectors with an evolutionarily conserved manner. Our data provide a mechanistic explanation of how arboviruses evolutionarily modulate host defenses for rapid transmission.

Interaction estimation of pathogenicity determinant protein ßC1 encoded by Cotton leaf curl Multan Betasatellite with Nicotiana benthamiana Nuclear Transport Factor 2.

Background: Begomovirus is one of the most devastating pathogens that can cause more than 90% yield loss in various crop plants. The pathogenicity determinant ßC1, located on the betasatellite associated with monopartite begomoviruses, alters the host signaling mechanism to enhance the viral disease phenotype by undermining the host immunity. The understanding of its interacting proteins in host plants to develop disease symptoms such as curly leaves, enations, vein swelling, and chlorosis is crucial to enhance the disease resistance in crop plants. The current study was designed to reveal the contribution of ßC1 in disease pathogenicity and to unveil potential interacting partners of ßC1 protein in the model plant Nicotiana benthamiana. Methods: The ßC1 gene was cloned in pGKBT7 and used as bait against the cDNA library of N. benthamiana and its pathogenesis was tested against the healthy plant and the plants infiltrated with empty vectors. The yeast two-hybrid-based screening was performed to find the interacting factors. Successful interacting proteins were screened and evaluated in various steps and confirmed by sequence analysis. The three-dimensional structure of the Nuclear Transport Factor 2 (NTF2) protein was predicted, and in-silico protein-protein interaction was evaluated. Furthermore, protein sequence alignment and molecular phylogenetic analysis were carried out to identify its homologues in other related families. In-silico analyses were performed to validate the binding affinity of ßC1 protein with NTF2. The 3D model was predicted by using I-TASSER and then analyzed by SWISS MODEL-Workspace, RAMPAGE, and Verify 3D. The interacting amino acid residues of ßC1 protein with NTF2 were identified by using PyMOL and Chimera. Results: The agroinfiltrated leaf samples developed severe phenotypic symptoms of virus infection. The yeast-two-hybrid study identified the NTF2 as a strong interacting partner of the ßC1. The NTF2 in Solanaceae and Nicotiana was found to be evolved from the Brassica and Gossypium species. The in-silico interaction studies showed a strong binding affinity with releasing energy value of -730.6 KJ/mol, and the involvement of 10 amino acids from the middle portion towards the C-terminus and five amino acid residues from the middle portion of ßC1 to interact with six amino acids of NTF2. The study not only provided an insight into the molecular mechanism of pathogenicity but also put the foundation stone to develop the resistance genotypes for commercial purposes and food security.

Development of transgenic okra (Abelmoschus esculentus L. Moench) lines having RNA mediated resistance to Yellow vein mosaic virus (Geminiviridae).

Begomovirus Yellow vein mosaic virus causes severe yield losses in okra and even the resistant lines developed through conventional breeding show susceptibility at various levels. This paper describes the development of YVMV resistant lines through RNAi strategy. A universal ihpRNA construct harbouring ßC1 ORF from the ß-satellite of the begomovirus was designed using pRNAi-LIC plasmid. Complementarity checks in sequence databases had shown no off-target effects by the target region and the success of siRNA in interference was proven using Custom Dicer-Substrate siRNA analysis. The ßC1 ORF of the begomovirus was PCR amplified and sequenced using the primer combination designed. The pRNAi-LIC vector, a derivative of pCAMBIA2300 containing duplicated CaMV 35S promoter and Nos terminator from pYL44, was SmaI digested and the amplified sense and antisense strands of the ßC1 region were cloned. E. coli transformed with the plasmid were screened for antibiotic resistance, and the plasmids confirmed for the sense and antisense regions through sequencing, were transferred to Agrobacterium tumefaciens strain GV3101. In planta transformation strategy was followed to transform a highly susceptible okra cv. Salkeerthi with ihpRNA-ßC1 cassette. Transformation success, confirmed by the amplification of sense strand using the primers VLIC1 and VLIC5, was 11.42 %. Transcription of siRNA from the ßC1 ORF in the transgenic lines was confirmed by its PCR amplification from the cDNA, using the stem loop primers designed (68 bp). When the transformed and healthy wild-type plants were co-grown with infected wild-type plants, inside an insect cage released with whiteflies and maintained within a containment facility, three of the four transgenic plants remained completely healthy throughout the crop span.

Loss of Function of mtHsp70 Chaperone Variants Leads to Mitochondrial Dysfunction in Congenital Sideroblastic Anemia.

Congenital Sideroblastic Anemias (CSA) is a group of rare genetic disorders characterized by the abnormal accumulation of iron in erythrocyte precursors. A common hallmark underlying these pathological conditions is mitochondrial dysfunction due to altered protein homeostasis, heme biosynthesis, and oxidative phosphorylation. A clinical study on congenital sideroblastic anemia has identified mutations in mitochondrial Hsp70 (mtHsp70/Mortalin). Mitochondrial Hsp70 plays a critical role in maintaining mitochondrial function by regulating several pathways, including protein import and folding, and iron-sulfur cluster synthesis. Owing to the structural and functional homology between human and yeast mtHsp70, we have utilized the yeast system to delineate the role of mtHsp70 variants in the etiology of CSA's. Analogous mutations in yeast mtHsp70 exhibited temperature-sensitive growth phenotypes under non-respiratory and respiratory conditions. In vivo analyses indicate a perturbation in mitochondrial mass and functionality accompanied by an alteration in the organelle network and cellular redox levels. Preliminary in vitro biochemical studies of mtHsp70 mutants suggest impaired import function, altered ATPase activity and substrate interaction. Together, our findings suggest the loss of chaperone activity to be a pivotal factor in the pathophysiology of congenital sideroblastic anemia.

Development of a LAMP assay using a portable device for the real-time detection of cotton leaf curl disease in field conditions.

Cotton production is seriously affected by the prevalent cotton leaf curl disease (CLCuD) that originated from Nigeria (Africa) to various parts of Asia including Pakistan, India, China and Philippines. Due to CLCuD, Pakistan suffers heavy losses approximately 2 billion USD per annum. Numerous reports showed that CLCuD is associated with multiple species of begomoviruses, alphasatellites and a single species of betasatellite, that is 'Cotton leaf curl Multan betasatellite' (CLCuMuB). The most prevalent form of CLCuD is the combination of 'Cotton leaf curl Kokhran virus'-Burewala strain (CLCuKoV-Bur) and CLCuMuB. Thus, the availability of an in-field assay for the timely detection of CLCuD is important for the control and management of the disease. In this study, a robust method using the loop-mediated isothermal amplification (LAMP) assay was developed for the detection of CLCuD. Multiple sets of six primers were designed based on the conserved regions of CLCuKoV-Bur and CLCuMuB-ßC1 genes. The results showed that the primer set targeting the CLCuMuB-ßC1 gene performed best when the LAMP assay was performed at 58°C using 100 ng of total plant tissue DNA as a template in a 25 µl reaction volume. The limit of detection for the assay was as low as 22 copies of total purified DNA template per reaction. This assay was further adapted to perform as a colorimetric and real-time LAMP assay which proved to be advantageously applied for the rapid and early point-of-care detection of CLCuD in the field. Application of the assay could help to prevent the huge economic losses caused by the disease and contribute to the socio-economic development of underdeveloped countries.

Begomovirus-Associated Betasatellite Virulence Factor ßC1 Attenuates Tobacco Defense to Whiteflies via Interacting With Plant SKP1.

Plant-mediated interactions between plant viruses and their vectors are important determinants of the population dynamics of both types of organisms in the field. The whitefly Bemisia tabaci can establish mutualism with begomoviruses via their shared host plants. This mutualism is achieved by the interaction between virulence factors and their host proteins. While the virulence factor ßC1 encoded by tomato yellow leaf curl China betasatellite (TYLCCNB), a subviral agent associated to the begomovirus tomato yellow leaf curl China virus (TYLCCNV), may interact with plant protein MYC2, thereby establishing the indirect mutualism between TYLCCNV and whitefly, whether other mechanisms are involved remains unknown. Here, we found the in vitro and in vivo interactions between ßC1 and tobacco protein S-phase kinase associated protein 1 (NtSKP1). Silencing the expression of NtSKP1 enhanced the survival rate and fecundity of whiteflies on tobacco plants. NtSKP1 could activate the transcription of genes in jasmonic acid (JA) pathways by impairing the stabilization of JAZ1 protein. Moreover, ßC1-NtSKP1 interaction could interfere JAZ1 degradation and attenuate the plant JA defense responses. These results revealed a novel mechanism underlying the better performance of whiteflies on TYLCCNV/TYLCCNB-infected plants.

Development of Virus Resistance Transgenic Cotton Using Cotton Leaf Curl Virus Antisense ßC1 Gene.

Cotton (Gossypium hirsutum L.) is the most economically important crop in the world and produced 90% of the total natural cellulose fiber which is utilized to make cotton fabrics. The production of cotton is affected by many several diseases, and among them, viral disease, especially leaf curl, is the most destructive disease caused by a begomovirus transmitted by whiteflies vector. Plant biotechnology has provided an opportunity to develop transgenic plant with variable traits against biotic and abiotic stress such as resistance against pathogens, yield, quality, and salinity. Transgenic cotton (Gossypium hirsutum L., cv. Coker 312) plants were raised against leaf curl disease using bC1 gene in antisense orientation through Agrobacterium-mediated transformation somatic embryogenesis system. In this chapter, a standardized protocol will be given to raise virus resistance transgenic cotton.

Multifaceted role of geminivirus associated betasatellite in pathogenesis.

Begomoviruses have emerged as a group of plant pathogens that cause devastating diseases in a wide range of crops in tropical and subtropical regions of the world. Betasatellites, the circular single-stranded DNA molecules with the size of almost half of that of the associated helper begomoviruses, are often essential for the production of typical disease symptoms in several virus-host systems. Association of betasatellites with begomoviruses results in more severe symptoms in the plants and affects the yield of numerous crops leading to huge agroeconomic losses. ßC1, the only protein encoded by betasatellites, plays a multifaceted role in the successful establishment of infection. This protein counteracts the innate defence mechanisms of the host, like RNA silencing, ubiquitin-proteasome system and defence responsive hormones. In the last two decades, the molecular aspect of betasatellite pathogenesis has attracted much attention from the researchers worldwide, and reports have shown that ßC1 protein aggravates the helper begomovirus disease complex by modulating specific host factors. This review discusses the molecular aspects of the pathogenesis of betasatellites, including various ßC1-host factor interactions and their effects on the suppression of defence responses of the plants.

Tomato Yellow Leaf Curl China Virus Impairs Photosynthesis in the Infected Nicotiana benthamiana with ßC1 as an Aggravating Factor.

Tomato yellow leaf curl China virus is a species of the widespread geminiviruses. The infection of Nicotiana benthamiana by Tomato yellow leaf curl China virus (TYLCCNV) causes a reduction in photosynthetic activity, which is part of the viral symptoms. ßC1 is a viral factor encoded by the betasatellite DNA (DNAß) accompanying TYLCCNV. It is a major viral pathogenicity factor of TYLCCNV. To elucidate the effect of ßC1 on plants' photosynthesis, we measured the relative chlorophyll (Chl) content and Chl fluorescence in TYLCCNV-infected and ßC1 transgenic N. benthamiana plants. The results showed that Chl content is reduced in TYLCCNV A-infected, TYLCCNV A plus DNAß (TYLCCNV A + ß)-infected and ßC1 transgenic plants. Further, changes in Chl fluorescence parameters, such as electron transport rate, F v /F m , NPQ, and qP, revealed that photosynthetic efficiency is compromised in the aforementioned N. benthamiana plants. The presense of ßC1 aggravated the decrease of Chl content and photosynthetic efficiency during viral infection. Additionally, the real-time quantitative PCR analysis of oxygen evolving complex genes in photosystem II, such as PsbO, PsbP, PsbQ, and PsbR, showed a significant reduction of the relative expression of these genes at the late stage of TYLCCNV A + ß infection and at the vegetative stage of ßC1 transgenic N. benthamiana plants. In summary, this study revealed the pathogenicity of TYLCCNV in photosynthesis and disclosed the effect of ßC1 in exacerbating the damage in photosynthesis efficiency by TYLCCNV infection.

An Insight into Cotton Leaf Curl Multan Betasatellite, the Most Important Component of Cotton Leaf Curl Disease Complex.

Cotton leaf curl disease (CLCuD) is one of the most economically important diseases and is a constraint to cotton production in major producers, Pakistan and India. CLCuD is caused by monopartite plant viruses belonging to the family Geminiviridae (genus Begomovirus), in association with an essential, disease-specific satellite, Cotton leaf curl Multan betasatellite (CLCuMuB) belonging to a newly-established family Tolecusatellitidae (genus Betasatellite). CLCuMuB has a small genome (ca. 1350 nt) with a satellite conserved region, an adenine-rich region and a single gene that encodes for a multifunctional ßC1 protein. CLCuMuB ßC1 protein has a major role in pathogenicity and symptom determination, and alters several host cellular functions like autophagy, ubiquitination, and suppression of gene silencing, to assist CLCuD infectivity. Efficient trans-replication ability of CLCuMuB with several monopartite and bipartite begomoviruses, is also associated with the rapid evolution and spread of CLCuMuB. In this article we comprehensively reviewed the role of CLCuMuB in CLCuD, focusing on the ßC1 functions and its interactions with host proteins.