Voltage-dependent anion channel proteins associate with dynamic Bamboo mosaic virus-induced complexes.

Infection cycles of viruses are highly dependent on membrane-associated host factors. To uncover the infection cycle of Bamboo mosaic virus (BaMV) in detail, we purified the membrane-associated viral complexes from infected Nicotiana benthamiana plants and analyzed the involved host factors. Four isoforms of voltage-dependent anion channel (VDAC) proteins on the outer membrane of mitochondria were identified due to their upregulated expression in the BaMV complex-enriched membranous fraction. Results from loss- and gain-of-function experiments indicated that NbVDAC2, -3, and -4 are essential for efficient BaMV accumulation. During BaMV infection, all NbVDACs concentrated into larger aggregates, which overlapped and trafficked with BaMV virions to the structure designated as the "dynamic BaMV-induced complex." Besides the endoplasmic reticulum and mitochondria, BaMV replicase and double-stranded RNAs were also found in this complex, suggesting the dynamic BaMV-induced complex is a replication complex. Yeast two-hybrid and pull-down assays confirmed that BaMV triple gene block protein 1 (TGBp1) could interact with NbVDACs. Confocal microscopy revealed that TGBp1 is sufficient to induce NbVDAC aggregates, which suggests that TGBp1 may play a pivotal role in the NbVDAC-virion complex. Collectively, these findings indicate that NbVDACs may associate with the dynamic BaMV-induced complex via TGBp1 and NbVDAC2, -3, or -4 and can promote BaMV accumulation. This study reveals the involvement of mitochondrial proteins in a viral complex and virus infection.

Identification of quantitative trait nucleotides and candidate genes for tuber yield and mosaic virus tolerance in an elite population of white guinea yam (Dioscorea rotundata) using genome-wide association scan.

BACKGROUND: Improvement of tuber yield and tolerance to viruses are priority objectives in white Guinea yam breeding programs. However, phenotypic selection for these traits is quite challenging due to phenotypic plasticity and cumbersome screening of phenotypic-induced variations. This study assessed quantitative trait nucleotides (QTNs) and the underlying candidate genes related to tuber yield per plant (TYP) and yam mosaic virus (YMV) tolerance in a panel of 406 white Guinea yam (Dioscorea rotundata) breeding lines using a genome-wide association study (GWAS). RESULTS: Population structure analysis using 5,581 SNPs differentiated the 406 genotypes into seven distinct sub-groups based delta K. Marker-trait association (MTA) analysis using the multi-locus linear model (mrMLM) identified seventeen QTN regions significant for TYP and five for YMV with various effects. The seveteen QTNs were detected on nine chromosomes, while the five QTNs were identified on five chromosomes. We identified variants responsible for predicting higher yield and low virus severity scores in the breeding panel through the marker-effect prediction. Gene annotation for the significant SNP loci identified several essential putative genes associated with the growth and development of tuber yield and those that code for tolerance to mosaic virus. CONCLUSION: Application of different multi-locus models of GWAS identified 22 QTNs. Our results provide valuable insight for marker validation and deployment for tuber yield and mosaic virus tolerance in white yam breeding. The information on SNP variants and genes from the present study would fast-track the application of genomics-informed selection decisions in breeding white Guinea yam for rapid introgression of the targeted traits through markers validation.

Construction of an infectious dahlia common mosaic virus clone.

Dahlia is a major ornamental plant that is cultivated worldwide. However, dahlia plants, which are mainly propagated through vegetative reproduction, are susceptible to widespread damage by viruses, and viral control requires that the nature of the infecting virus(es) be known. In this study, dahlia common mosaic virus (DCMV) was detected for the first time in Japan and sequenced. This is the first report of an infectious DCMV clone being constructed, and it will aid in the characterization of DCMV.

Marker-assisted selection complements phenotypic screening at seedling stage to identify cassava mosaic disease-resistant genotypes in African cassava populations.

Cassava mosaic disease (CMD) is a serious threat to cassava production in sub-Saharan Africa. The use of genomic-assisted selection at the seedling trial stage would help to reduce the time for release, breeding cost, and resources used, hence increase selection efficiency in cassava breeding programs. Five cassava populations were screened for resistance to CMD during the seedling evaluation trial at 1, 3, and 5 months after planting using a scale of 1-5. The genotypes in the five populations were also screened using six molecular markers linked to the CMD2 gene. The correlation between the phenotypic and marker data was estimated. Based on Cassava Mosaic Disease Severity Score (CMDSS), between 53 and 82% of the progenies were resistant across the populations with an average of 70.5%. About 70% of the progenies were identified to be resistant to the disease across the populations with a range of 62-80% using the marker data. With both marker data and CMDSS combined, 40-60% of the progenies in each population, with an average of 52%, were identified to be resistant to CMD. There was a fairly significant correlation between the marker data and CMDSS in each cassava population with correlation coefficients ranging from 0.2024 to 0.3460 suggesting that novel genes not associated to the markers used might be involved in the resistance to CMD. The resistant genotypes identified in this study with potential for other desirable traits were selected for evaluation at the advanced trial stage thereby shortening the period required for the breeding program.

siRNA biogenesis and advances in topically applied dsRNA for controlling virus infections in tomato plants.

A non-transgenic approach based on RNA interference was employed to induce protection against tomato mosaic virus (ToMV) infection in tomato plants. dsRNA molecules targeting the cp gene of ToMV were topically applied on plants prior to virus inoculation. Protection was dose-dependent and sequence-specific. While no protection was achieved when 0-16 µg dsRNA were used, maximum rates of resistance (60 and 63%) were observed in doses of 200 and 400 µg/plant, respectively. Similar rates were also obtained against potato virus Y when targeting its cp gene. The protection was quickly activated upon dsRNA application and lasted for up to 4 days. In contrast, no detectable antiviral response was triggered by the dsRNA from a begomovirus genome, suggesting the method is not effective against phloem-limited DNA viruses. Deep sequencing was performed to analyze the biogenesis of siRNA populations. Although long-dsRNA remained in the treated leaves for at least 10 days, its systemic movement was not observed. Conversely, dsRNA-derived siRNA populations (mainly 21- and 22-nt) were detected in non-treated leaves, which indicates endogenous processing and transport through the plant. Altogether, this study provides critical information for the development of novel tools against plant viruses; strengths and limitations inherent to the systems are discussed.

Physiological and molecular mechanisms governing the effect of virus-free chewing cane seedlings on yield and quality.

The effects of increasing yield and quality of virus-free chewing cane seedlings and their physiological and molecular basis were studied in this study. Results showed that compared with infected seedlings (the control), the yield of chewing cane stems grown from virus-free seedlings increased by 21.81-29.93%, stem length increased by 28.66-34.49 cm, internode length increased by 2.16-2.68 cm, the single stem weight increased by 20.10-27.68%, the reducing sugar increased by 0.91-1.15% (absolute value), and sucrose increased by - 0.06-1.33% (absolute value). The decrease in sucrose content did not reach significant level, but all other parameters were reached significant level. The chlorophyll content, photosynthetic parameters such as stomatal conductance (Gs), net photosynthetic rate (Pn) and transpiration rate (Tr), the activity of photosynthetic key enzymes ribulose-1,5-bisphosphate carboxylase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC), and gene (pepc, rbcS, and rbcL) expression levels were all greater in virus-free seedlings than infected seedlings. The content of superoxide anion (O2-) and malondialdehyde (MDA) in virus-free seedlings was lower than infected seedlings at the main growth stage. With increased development, the activities of the antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were gradually higher in virus-free seedlings than infected seedlings. Our results indicate that virus-free seedlings may improve photosynthesis efficiency and promote photosynthesis by increasing chlorophyll content, photosynthetic key enzyme activity, and the gene expression levels in leaves. By increasing the activity of antioxidant enzymes, reducing the degree of membrane lipid peroxidation, and improving the stress resistance of chewing cane, the virus-free chewing cane seedlings increased yield and quality. Our findings provide a scientific and theoretical basis for the promotion and application of virus-free chewing cane seedlings.

Identification and Characterization of NPR1 and PR1 Homologs in Cymbidium orchids in Response to Multiple Hormones, Salinity and Viral Stresses.

The plant nonexpressor of pathogenesis-related 1 (NPR1) and pathogenesis-associated 1 (PR1) genes play fundamental roles in plant immunity response, as well as abiotic-stress tolerance. Nevertheless, comprehensive identification and characterization of NPR1 and PR1 homologs has not been conducted to date in Cymbidium orchids, a valuable industrial crop cultivated as ornamental and medicinal plants worldwide. Herein, three NPR1-like (referred to as CsNPR1-1, CsNPR1-2, and CsNPR1-3) and two PR1-like (CsPR1-1 and CsPR1-2) genes were genome-widely identified from Cymbidium orchids. Sequence and phylogenetic analysis revealed that CsNPR1-1 and CsNPR1-2 were grouped closest to NPR1 homologs in Zea mays (sharing 81.98% identity) and Phalaenopsis (64.14%), while CsNPR1-3 was classified into a distinct group with Oryza sativa NPR 3 (57.72%). CsPR1-1 and CsPR1-2 were both grouped closest to Phalaenopsis PR1 and other monocot plants. Expression profiling showed that CsNPR1 and CsPR1 were highly expressed in stem/pseudobulb and/or flower. Salicylic acid (SA) and hydrogen peroxide (H2O2) significantly up-regulated expressions of CsNPR1-2, CsPR1-1 and CsPR1-2, while CsNPR1-3, CsPR1-1 and CsPR1-2 were significantly up-regulated by abscisic acid (ABA) or salinity (NaCl) stress. In vitro transcripts of entire Cymbidium mosaic virus (CymMV) genomic RNA were successfully transfected into Cymbidium protoplasts, and the CymMV infection up-regulated the expression of CsNPR1-2, CsPR1-1 and CsPR1-2. Additionally, these genes were transiently expressed in Cymbidium protoplasts for subcellular localization analysis, and the presence of SA led to the nuclear translocation of the CsNPR1-2 protein, and the transient expression of CsNPR1-2 greatly enhanced the expression of CsPR1-1 and CsPR1-2. Collectively, the CsNPR1-2-mediated signaling pathway is SA-dependent, and confers to the defense against CymMV infection in Cymbidium orchids.

Plant virus interaction mechanism and associated pathways in mosaic disease of small cardamom (Elettaria cardamomum Maton) by RNA-Seq approach.

Small cardamom (Elettaria cardamomum), grown in limited coastal tropical countries is one of the costliest and widely exported agri-produce having global turnover of >10 billion USD. Mosaic/marble disease is one of the major impediments that requires understanding of disease at molecular level. Neither whole genome sequence nor any genomic resources are available, thus RNA seq approach can be a rapid and economical alternative. De novo transcriptome assembly was done with Illumina Hiseq data. A total of 5317 DEGs, 2267 TFs, 114 pathways and 175,952 genic region putative markers were obtained. Gene regulatory network analysis deciphered molecular events involved in marble disease. This is the first transcriptomic report revealing disease mechanism mediated by perturbation in auxin homeostasis and ethylene signalling leading to senescence. The web-genomic resource (SCMVTDb) catalogues putative molecular markers, candidate genes and transcript information. SCMVTDb can be used in germplasm improvement against mosaic disease in endeavour of small cardamom productivity. Availability of genomic resource, SCMVTDb: http://webtom.cabgrid.res.in/scmvtdb/.

Rice Stripe Mosaic Virus-Encoded P4 Is a Weak Suppressor of Viral RNA Silencing and Is Required for Disease Symptom Development.

Viral suppressors of RNA silencing (VSRs) are a cluster of viral proteins that have evolved to counteract eukaryotic antiviral RNA silencing pathways, thereby contributing to viral pathogenicity. In this study, we revealed that the matrix protein P4 encoded by rice stripe mosaic virus (RSMV), which is an emerging cytoplasmic rhabdovirus, is a weak RNA silencing suppressor. By conducting yeast two-hybrid, bimolecular fluorescence complementation, and subcellular colocalization assays, we proved that P4 interacts with the rice endogenous suppressor of gene silencing 3 (OsSGS3). We also determined that P4 overexpression has no effect on OsSGS3 transcription. However, P4 can promote the degradation of OsSGS3 via ubiquitination and autophagy. Additionally, a potato virus X-based expression system was used to confirm that P4 enhances the development of mosaic symptoms on Nicotiana benthamiana leaves by promoting hydrogen peroxide accumulation but not cell death. To verify whether P4 is a pathogenicity factor in host plants, we generated transgenic P4-overexpressing rice plants that exhibited disease-related developmental defects including decreased plant height and excessive tillering. Our data suggest that RSMV-encoded P4 serves as a weak VSR that inhibits antiviral RNA silencing by targeting OsSGS3.

Characterization of Maize miRNAs in Response to Synergistic Infection of Maize Chlorotic Mottle Virus and Sugarcane Mosaic Virus.

The synergistic infection of maize chlorotic mottle virus (MCMV) and sugarcane mosaic virus (SCMV) causes maize lethal necrosis, with considerable losses to global maize production. microRNAs (miRNAs) are conserved non-coding small RNAs that play essential regulatory roles in plant development and environmental stress responses, including virus infection. However, the characterization of maize miRNAs in response to synergistic infection of MCMV and SCMV remains largely unknown. In this study, the profiles of small RNAs from MCMV and SCMV single- and co-infected (S + M) maize plants were obtained by high-throughput sequencing. A total of 173 known miRNAs, belonging to 26 miRNA families, and 49 novel miRNAs were profiled. The expression patterns of most miRNAs in S + M-infected maize plants were similar to that in SCMV-infected maize plants, probably due to the existence of RNA silencing suppressor HC-Pro. Northern blotting and quantitative real-time PCR were performed to validate the accumulation of miRNAs and their targets in different experimental treatments, respectively. The down-regulation of miR159, miR393, and miR394 might be involved in antiviral defense to synergistic infection. These results provide novel insights into the regulatory networks of miRNAs in maize plants in response to the synergistic infection of MCMV and SCMV.

P7 and P8 proteins of High Plains wheat mosaic virus, a negative-strand RNA virus, employ distinct mechanisms of RNA silencing suppression.

High Plains wheat mosaic virus (genus Emaravirus), an octapartite negative-sense RNA virus, encodes two RNA silencing suppressors, P7 and P8. In this study, we found that P7 and P8 efficiently delayed the onset of dsRNA-induced transitive pathway of RNA silencing. Electrophoretic mobility shift assays (EMSA) revealed that only P7 protected long dsRNAs from dicing in vitro and bound weakly to 21- and 24-nt PTGS-like ds-siRNAs. In contrast, P8 bound strongly and relatively weakly to 21- and 24-nt ds-siRNAs, respectively, suggesting size-specific binding. In EMSA, neither protein bound to 180-nt and 21-nt ssRNAs at detectable levels. Sequence analysis revealed that P7 contains a conserved GW motif. Mutational disruption of this motif resulted in loss of suppression of RNA silencing and pathogenicity enhancement, and failure to complement the silencing suppression-deficient wheat streak mosaic virus. Collectively, these data suggest that P7 and P8 proteins utilize distinct mechanisms to overcome host RNA silencing for successful establishment of systemic infection in planta.

Triticum aestivum heat shock protein 23.6 interacts with the coat protein of wheat yellow mosaic virus.

The role of heat shock proteins (HSPs) in viral replication has been described in numerous publications. Wheat yellow mosaic virus (WYMV) belongs to the genus Bymovirus (family Potyviridae), which causes yellow mosaic and dwarf symptoms in wheat (Triticum aestivum). In this study, the T. aestivum heat shock protein 23.6 (TaHSP23.6), which belongs to the small heat shock protein family, was shown to interact with the WYMV coat protein (CP) in a yeast two-hybrid screen. The co-localization and interaction between TaHSP23.6 and WYMV CP were additionally verified in Nicotiana benthamiana by co-localization assays and bimolecular fluorescence complementation (BiFC). Not only the transcription of TaHSP23.6 but also that of other HSP family members (TaHSP70, TaHSP90, and TaHSP101) was up-regulated in WYMV-infected leaves, as shown by semi-quantitative PCR assays. Interestingly, the expression levels of the T. aestivum heat stress transcription factor A2 (TaHSFA2) members were varied in response to WYMV infection. Thus, our results provide insights into the interaction between TaHSP23.6 and WYMV infection.

Octapartite negative-sense RNA genome of High Plains wheat mosaic virus encodes two suppressors of RNA silencing.

High Plains wheat mosaic virus (HPWMoV, genus Emaravirus; family Fimoviridae), transmitted by the wheat curl mite (Aceria tosichella Keifer), harbors a monocistronic octapartite single-stranded negative-sense RNA genome. In this study, putative proteins encoded by HPWMoV genomic RNAs 2-8 were screened for potential RNA silencing suppression activity by using a green fluorescent protein-based reporter agroinfiltration assay. We found that proteins encoded by RNAs 7 (P7) and 8 (P8) suppressed silencing induced by single- or double-stranded RNAs and efficiently suppressed the transitive pathway of RNA silencing. Additionally, a Wheat streak mosaic virus (WSMV, genus Tritimovirus; family Potyviridae) mutant lacking the suppressor of RNA silencing (ΔP1) but having either P7 or P8 from HPWMoV restored cell-to-cell and long-distance movement in wheat, thus indicating that P7 or P8 rescued silencing suppressor-deficient WSMV. Furthermore, HPWMoV P7 and P8 substantially enhanced the pathogenicity of Potato virus X in Nicotiana benthamiana. Collectively, these data demonstrate that the octapartite genome of HPWMoV encodes two suppressors of RNA silencing.

Cleaning cassava genotypes infected with cassava frogskin disease via in vitro shoot tip culture.

This study aimed to develop a methodology for eliminating cassava frogskin disease (CFSD) from in vitro shoot tip culture by associating thermotherapy and tetracycline. Cuttings from different accessions (BGM0232, BGM0315, BGM0464, BGM584, BGM0841, and BGM1342), infected with CFSD according to visual inspection of the disease symptoms, were used for cleaning. To verify the absence of other diseases, the plants were indexed for Cassava common mosaic virus - CsCMV (by ELISA) and Cassava vein mosaic virus - CsVMV (by polymerase chain reaction, PCR), proving that the accessions were free of these viruses, except for BGM0315 and BGM0464, which were infected with CsVMV. Subsequently, the cuttings were submitted to different tetracycline concentrations for 3 min, and then subjected to thermotherapy under different temperatures (35°, 38°, 40°, 45°, and 55°C). Shoots of 2 cm were harvested, and their surfaces were sterilized in a laminar flow chamber. Subsequently, the shoot tips of different sizes were removed (0.2, 0.4, 0.5, and 1.0 mm) for inoculation in a culture medium with tetracycline at the same concentrations in which the cuttings were dipped. After 60 days of cultivation, the explants were transferred to a multiplication medium without antibiotics. Thirty days after the transfer, the viability of the regenerated plants was evaluated, which were then acclimatized for 70 days in a greenhouse and transferred to the field. After 7 months, a visual analysis of the symptomatic roots and a PCR analysis were held to prove the elimination of CFSD and CsVMV from the accessions infected with these viruses (BGM0315 and BGM0464), respectively. Most of the treatments resulted in 100% cleaning of CFSD-infected plants. From accessions that were also infected with CsVMV, only 2% of the plants remained infected, also demonstrating the cleaning efficiency of this protocol for this disease.

Cassava Mosaic and Brown Streak Diseases: Current Perspectives and Beyond.

Cassava is the fourth largest source of calories in the world but is subject to economically important yield losses due to viral diseases, including cassava brown streak disease and cassava mosaic disease. Cassava mosaic disease occurs in sub-Saharan Africa and the Asian subcontinent and is associated with nine begomovirus species, whereas cassava brown streak disease has to date been reported only in sub-Saharan Africa and is caused by two distinct ipomovirus species. We present an overview of key milestones and their significance in the understanding and characterization of these two major diseases as well as their associated viruses and whitefly vector. New biotechnologies offer a wide range of opportunities to reduce virus-associated yield losses in cassava for farmers and can additionally enable the exploitation of this valuable crop for industrial purposes. This review explores established and new technologies for genetic manipulation to achieve desired traits such as virus resistance.

Synergistic infection of BrYV and PEMV 2 increases the accumulations of both BrYV and BrYV-derived siRNAs in Nicotiana benthamiana.

Viral synergism is caused by co-infection of two unrelated viruses, leading to more severe symptoms or increased titres of one or both viruses. Synergistic infection of phloem-restricted poleroviruses and umbraviruses has destructive effects on crop plants. The mechanism underlying this synergy remains elusive. In our study, synergism was observed in co-infections of a polerovirus Brassica yellows virus (BrYV) and an umbravirus Pea enation mosaic virus 2 (PEMV 2) on Nicotiana benthamiana, which led to (1) increased titres of BrYV, (2) appearance of severe symptoms, (3) gain of mechanical transmission capacity of BrYV, (4) broader distribution of BrYV to non-vascular tissues. Besides, profiles of virus-derived small interfering RNAs (vsiRNAs) from BrYV and PEMV 2 in singly and doubly infected plants were obtained by small RNA deep sequencing. Our results showed that accumulation of BrYV vsiRNAs increased tremendously and ratio of positive to negative strand BrYV vsiRNAs differed between singly infected and co-infected plants. Positions to which the BrYV vsiRNAs mapped to the viral genome varied considerably during synergistic infection. Moreover, target genes of vsiRNAs were predicted and annotated. Our results revealed the synergistic characteristics during co-infection of BrYV and PEMV 2, and implied possible effects of synergism have on vsiRNAs.

The Potyviral P3 Protein Targets Eukaryotic Elongation Factor 1A to Promote the Unfolded Protein Response and Viral Pathogenesis.

The biochemical function of the potyviral P3 protein is not known, although it is known to regulate virus replication, movement, and pathogenesis. We show that P3, the putative virulence determinant of soybean mosaic virus (SMV), targets a component of the translation elongation complex in soybean. Eukaryotic elongation factor 1A (eEF1A), a well-known host factor in viral pathogenesis, is essential for SMV virulence and the associated unfolded protein response (UPR). Silencing GmEF1A inhibits accumulation of SMV and another ER-associated virus in soybean. Conversely, endoplasmic reticulum (ER) stress-inducing chemicals promote SMV accumulation in wild-type, but not GmEF1A-knockdown, plants. Knockdown of genes encoding the eEF1B isoform, which is important for eEF1A function in translation elongation, has similar effects on UPR and SMV resistance, suggesting a link to translation elongation. P3 and GmEF1A promote each other's nuclear localization, similar to the nuclear-cytoplasmic transport of eEF1A by the Human immunodeficiency virus 1 Nef protein. Our results suggest that P3 targets host elongation factors resulting in UPR, which in turn facilitates SMV replication and place eEF1A upstream of BiP in the ER stress response during pathogen infection.

Two Novel DNAs That Enhance Symptoms and Overcome CMD2 Resistance to Cassava Mosaic Disease.

UNLABELLED: Cassava mosaic begomoviruses (CMBs) cause cassava mosaic disease (CMD) across Africa and the Indian subcontinent. Like all members of the geminivirus family, CMBs have small, circular single-stranded DNA genomes. We report here the discovery of two novel DNA sequences, designated SEGS-1 and SEGS-2 (forsequencesenhancinggeminivirussymptoms), that enhance symptoms and break resistance to CMD. The SEGS are characterized by GC-rich regions and the absence of long open reading frames. Both SEGS enhanced CMD symptoms in cassava (Manihot esculentaCrantz) when coinoculated withAfrican cassava mosaic virus(ACMV),East African cassava mosaic Cameroon virus(EACMCV), orEast African cassava mosaic virus-Uganda(EACMV-UG). SEGS-1 also overcame resistance of a cassava landrace carrying the CMD2 resistance locus when coinoculated with EACMV-UG. Episomal forms of both SEGS were detected in CMB-infected cassava but not in healthy cassava. SEGS-2 episomes were also found in virions and whiteflies. SEGS-1 has no homology to geminiviruses or their associated satellites, but the cassava genome contains a sequence that is 99% identical to full-length SEGS-1. The cassava genome also includes three sequences with 84 to 89% identity to SEGS-2 that together encompass all of SEGS-2 except for a 52-bp region, which includes the episomal junction and a 26-bp sequence related to alphasatellite replication origins. These results suggest that SEGS-1 is derived from the cassava genome and facilitates CMB infection as an integrated copy and/or an episome, while SEGS-2 was originally from the cassava genome but now is encapsidated into virions and transmitted as an episome by whiteflies. IMPORTANCE: Cassava is a major crop in the developing world, with its production in Africa being second only to maize. CMD is one of the most important diseases of cassava and a serious constraint to production across Africa. CMD2 is a major CMD resistance locus that has been deployed in many cassava cultivars through large-scale breeding programs. In recent years, severe, atypical CMD symptoms have been observed occasionally on resistant cultivars, some of which carry the CMD2 locus, in African fields. In this report, we identified and characterized two DNA sequences, SEGS-1 and SEGS-2, which produce similar symptoms when coinoculated with cassava mosaic begomoviruses onto a susceptible cultivar or a CMD2-resistant landrace. The ability of SEGS-1 to overcome CMD2 resistance and the transmission of SEGS-2 by whiteflies has major implications for the long-term durability of CMD2 resistance and underscore the need for alternative sources of resistance in cassava.

Identification of haplotypes at the Rsv4 genomic region in soybean associated with durable resistance to soybean mosaic virus.

KEY MESSAGE: Discovery of new germplasm sources and identification of haplotypes for the durable Soybean mosaic virus resistance gene, Rsv 4, provide novel resources for map-based cloning and genetic improvement efforts in soybean. The Soybean mosaic virus (SMV) resistance locus Rsv4 is of interest because it provides a durable type of resistance in soybean [Glycine max (L.) Merr.]. To better understand its molecular basis, we used a population of 309 BC3F2 individuals to fine-map Rsv4 to a ~120 kb interval and leveraged this genetic information in a second study to identify accessions 'Haman' and 'Ilpumgeomjeong' as new sources of Rsv4. These two accessions along with three other Rsv4 and 14 rsv4 accessions were used to examine the patterns of nucleotide diversity at the Rsv4 region based on high-depth resequencing data. Through a targeted association analysis of these 19 accessions within the ~120 kb interval, a cluster of four intergenic single-nucleotide polymorphisms (SNPs) was found to perfectly associate with SMV resistance. Interestingly, this ~120 kb interval did not contain any genes similar to previously characterized dominant disease resistance genes. Therefore, a haplotype analysis was used to further resolve the association signal to a ~94 kb region, which also resulted in the identification of at least two Rsv4 haplotypes. A haplotype phylogenetic analysis of this region suggests that the Rsv4 locus in G. max is recently introgressed from G. soja. This integrated study provides a strong foundation for efforts focused on the cloning of this durable virus resistance gene and marker-assisted selection of Rsv4-mediated SMV resistance in soybean breeding programs.

Ultrastructural insights into tomato infections caused by three different pathotypes of Pepino mosaic virus and immunolocalization of viral coat proteins.

This paper presents studies on an ultrastructural analysis of plant tissue infected with different pathotypes of Pepino mosaic virus (PepMV) and the immunolocalization of viral coat proteins. Because the PepMV virus replicates with a high mutation rate and exhibits significant genetic diversity, therefore, isolates of PepMV display a wide range of symptoms on infected plants. In this work, tomato plants of the Beta Lux cultivar were inoculated mechanically with three pathotypes representing the Chilean 2 (CH2) genotype: mild (PepMV-P22), necrotic (PepMV-P19) and yellowing (PepMV-P5-IY). The presence of viral particles in all infected plants in the different compartments of various cell types (i.e. spongy and palisade mesophyll, sieve elements and xylem vessels) was revealed via ultrastructural analyses. For the first time, it was possible to demonstrate the presence of crystalline inclusions, composed of virus-like particles. In the later stage of PepMV infection (14 dpi) various pathotype-dependent changes in the structure of the individual organelles (i.e. mitochondria, chloroplasts) were found. The strongest immunogold labeling of the viral coat proteins was also observed in plants infected by necrotic isolates. A large number of viral coat proteins were marked in the plant conductive elements, both xylem and phloem.