The prominent multiplication of Japanese soil-borne wheat mosaic virus co-infected with barley yellow mosaic virus in barley.

Various members of the viral genera Furovirus and Bymovirus are damaging pathogens of a range of crop species. Infection of the soil-borne plasmodiophorid Polymyxa graminis transmits both Japanese soil-borne wheat mosaic virus (JSBWMV) and the barley yellow mosaic virus (BaYMV) to barley, but their interaction during an episode of their co-infection has not been characterized to date. Here, we present an analysis of the titer of JSBWMV and BaYMV in plants of winter barley growing over a five-month period from late fall until mid-spring. Although JSBWMV was detectable in the plants' roots four weeks earlier than BaYMV, the translocation of both viruses from the root to the leaves occurred nearly simultaneously. Both viruses were co-localized in the roots, leaf sheathes, and leaf blades; however, in some stripes of leaf veins where infection by JSBWMV was prominent, BaYMV was not detectable. A substantial titer of both viruses persisted until early spring, after which JSBWMV became more prominent, being in a range of 10 to 100 times abundant of BaYMV. However, JSBWMV was only able to infect a single wheat accession (cv. Norin 61), whereas all of the wheat entries assayed appeared to be immune to BaYMV infection. Overall, our findings highlight the importance of resistance mechanisms against soil-borne viruses in cereal crops, expanding our understanding of plant-virus interactions and potentially informing strategies for crop protection against viral pathogens.

Identification of novel QTL contributing to barley yellow mosaic resistance in wild barley (Hordeum vulgare spp. spontaneum).

BACKGROUND: Barley yellow mosaic disease (BYMD) caused by Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV) seriously threatens the production of winter barley. Cultivating and promoting varieties that carry disease-resistant genes is one of the most powerful ways to minimize the disease's effect on yield. However, as the BYMD virus mutates rapidly, resistance conferred by the two cloned R genes to the virus had been overcome by new virus strains. There is an urgent need for novel resistance genes in barley that convey sustainable resistance to newly emerging virus strains causing BYMD. RESULTS: A doubled haploid (DH) population derived from a cross of SRY01 (BYMD resistant wild barley) and Gairdner (BYMD susceptible barley cultivar) was used to explore for QTL of resistance to BYMD in barley. A total of six quantitative trait loci (qRYM-1H, qRYM-2Ha, qRYM-2Hb, qRYM-3H, qRYM-5H, and qRYM-7H) related to BYMD resistance were detected, which were located on chromosomes 1H, 2H, 3H, 5H, and 7H. Both qRYM-1H and qRYM-2Ha were detected in all environments. qRYM-1H was found to be overlapped with rym7, a known R gene to the disease, whereas qRYM-2Ha is a novel QTL on chromosome 2H originated from SRY01, explaining phenotypic variation from 9.8 to 17.8%. The closely linked InDel markers for qRYM-2Ha were developed which could be used for marker-assisted selection in barley breeding. qRYM-2Hb and qRYM-3H were stable QTL for specific resistance to Yancheng and Yangzhou virus strains, respectively. qRYM-5H and qRYM-7H identified in Yangzhou were originated from Gairdner. CONCLUSIONS: Our work is focusing on a virus disease (barley yellow mosaic) of barley. It is the first report on BYMD-resistant QTL from wild barley accessions. One novel major QTL (qRYM-2Ha) for the resistance was detected. The consistently detected new genes will potentially serve as novel sources for achieving pre-breeding barley materials with resistance to BYMD.

Genomic prediction models trained with historical records enable populating the German ex situ genebank bio-digital resource center of barley (Hordeum sp.) with information on resistances to soilborne barley mosaic viruses.

KEY MESSAGE: Genomic prediction with special weight of major genes is a valuable tool to populate bio-digital resource centers. Phenotypic information of crop genetic resources is a prerequisite for an informed selection that aims to broaden the genetic base of the elite breeding pools. We investigated the potential of genomic prediction based on historical screening data of plant responses against the Barley yellow mosaic viruses for populating the bio-digital resource center of barley. Our study includes dense marker data for 3838 accessions of winter barley, and historical screening data of 1751 accessions for Barley yellow mosaic virus (BaYMV) and of 1771 accessions for Barley mild mosaic virus (BaMMV). Linear mixed models were fitted by considering combinations for the effects of genotypes, years, and locations. The best linear unbiased estimations displayed a broad spectrum of plant responses against BaYMV and BaMMV. Prediction abilities, computed as correlations between predictions and observed phenotypes of accessions, were low for the marker-assisted selection approach amounting to 0.42. In contrast, prediction abilities of genomic best linear unbiased predictions were high, with values of 0.62 for BaYMV and 0.64 for BaMMV. Prediction abilities of genomic prediction were improved by up to ~ 5% using W-BLUP, in which more weight is given to markers with significant major effects found by association mapping. Our results outline the utility of historical screening data and W-BLUP model to predict the performance of the non-phenotyped individuals in genebank collections. The presented strategy can be considered as part of the different approaches used in genebank genomics to valorize genetic resources for their usage in disease resistance breeding and research.

PAL1 gene of the phenylpropanoid pathway increases resistance to the Cassava brown streak virus in cassava.

BACKGROUND: The phenylalanine ammonia lyase genes play crucial role in plant response to biotic and abiotic stresses. In this study, we characterized the role of PAL genes in increasing resistance to the Cassava brown streak virus that causes the economically important cassava brown streak disease (CBSD) on cassava in Africa. METHODS: The whole transcriptomes of eight cassava varieties differing in resistance to CBSD were obtained at 1, 5 and 8 weeks after CBSV infection. RESULTS: Analysis of RNA-Seq data identified the overexpression of PAL1, PAL2, cinnamic acid and two chalcone synthase genes in CBSD-resistant cassava varieties, which was subsequently confirmed by RT-qPCR. The exogenous application of Acibenzolar-S-Methyl induced PAL1 gene expression to enhance resistance in the susceptible var. Kalawe. In contrast, the silencing of PAL1 by RNA interference led to increased susceptibility of the resistant var. Kaleso to CBSD. CONCLUSIONS: PAL1 gene of the phenylpropanoid pathway has a major role in inducing resistance to CBSD in cassava plants and its early induction is key for CBSD resistance.

Estimating epidemiological parameters from experiments in vector access to host plants, the method of matching gradients.

Estimation of pathogenic life-history values, for instance the duration a pathogen is retained in an insect vector (i.e., retention period) is of particular importance for understanding plant disease epidemiology. How can we extract values for these epidemiological parameters from conventional small-scale laboratory experiments in which transmission success is measured in relation to durations of vector access to host plants? We provide a solution to this problem by deriving formulae for the empirical curves that these experiments produce, called access period response curves (i.e., transmission success vs access period). We do this by writing simple equations for the fundamental life-cycle components of insect vectors in the laboratory. We then infer values of epidemiological parameters by matching the theoretical and empirical gradients of access period response curves. Using the example of Cassava brown streak virus (CBSV), which has emerged in sub-Saharan Africa and now threatens regional food security, we illustrate the method of matching gradients. We show how applying the method to published data produces a new understanding of CBSV through the inference of retention period, acquisition period and inoculation period parameters. We found that CBSV is retained for a far shorter duration in its insect vector (Bemisia tabaci whitefly) than had previously been assumed. Our results shed light on a number of critical factors that may be responsible for the transition of CBSV from sub- to super-threshold R0 in sub-Saharan Africa. The method is applicable to plant pathogens in general, to supply epidemiological parameter estimates that are crucial for practical management of epidemics and prediction of pandemic risk.

Bulked segregant RNA-sequencing (BSR-seq) identified a novel rare allele of eIF4E effective against multiple isolates of BaYMV/BaMMV.

KEY MESSAGE: A novel rare allele of the barley host factor gene eIF4E for BaMMV/BaYMV infection was identified in an Iranian landrace that showed broad resistance to barley yellow mosaic virus disease, and molecular markers facilitating efficient selection were developed. The soil-borne yellow mosaic virus disease caused by different strains of barley yellow mosaic virus (BaYMV) and barley mild mosaic virus (BaMMV) is a major threat to winter barley (Hordeum vulgare) production in Europe and East Asia. However, the exploration of resistant germplasm or casual genes for barley breeding is rather limited in relation to the rapid diversification of viral strains. Here, we identified an Iranian barley landrace 'HOR3298,' which represented complete resistance to BaYMV and BaMMV. In contrast to rym4 and rym5, which act as the predominant source in Europe and East Asia for breeding resistant cultivars over decades and which have been overcome by several virulent isolates, this landrace showed broad-spectrum resistance to multiple isolates of BaYMV/BaMMV in the fields of Germany and China. By employment of bulked segregant RNA sequencing, test for allelism, and haplotype analysis, a recessive resistance gene in 'HOR3298' was genetically mapped coincident with the host factor eukaryotic translation initiation factor 4E (eIF4E, causal gene of rym4 and rym5). The eIF4EHOR3298 allele encoded for a novel haplotype that contained an exclusive nucleotide mutation (G565A) in the coding sequence. The easily handled markers were developed based on the exclusively rare variation, providing precise selection of this allele. Thus, this work provided a novel reliable resistance source and the feasible marker-assisted selection assays that can be used in breeding for barley yellow mosaic virus disease resistance in cultivated barley.

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.

Utilization of infectious clones to visualize Cassava brown streak virus replication in planta and gain insights into symptom development.

Cassava brown streak disease (CBSD) is a leading cause of cassava yield losses across eastern and central Africa and is having a severe impact on food security across the region. Despite its importance, relatively little is known about the mechanisms behind CBSD viral infections. We have recently reported the construction of Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) infectious clones (IC), which can be used to gain insights into the functions of viral proteins and sequences associated with symptom development. In this study, we perform the first reporter gene tagging of a CBSV IC, with the insertion of green fluorescent protein (GFP) sequence at two different genome positions. Nicotiana benthamiana infections with the CBSV_GFP ICs revealed active CBSV replication in inoculated leaves at 2-5 days post inoculation (dpi) and systemic leaves at 10-14 dpi. We also constructed the chimera CBSV_UCP IC, consisting of the CBSV genome with a UCBSV coat protein (CP) sequence replacement. N. benthamiana infections with CBSV_UCP revealed that the CBSV CP may be associated with high levels of viral accumulation and necrosis development during early infection. These initial manipulations pave the way for U/CBSV ICs to be used to understand U/CBSV biology that will inform vital CBSD control strategies.

Areca Palm Necrotic Ringspot Virus, Classified Within a Recently Proposed Genus Arepavirus of the Family Potyviridae, Is Associated With Necrotic Ringspot Disease in Areca Palm.

Areca palm (Areca catechu), one of the two most important commercial crops in Hainan, China, has been severely damaged by a variety of pathogens and insects. Here, we report a new disease, tentatively referred to as areca palm necrotic ringspot disease (ANRSD), which is highly epidemic in the main growing regions in Hainan. Transmission electron microscopy observation and small RNA deep sequencing revealed the existence of a viral agent of the family Potyviridae in a diseased areca palm plant (XC1). The virus was tentatively named areca palm necrotic ringspot virus (ANRSV). Subsequently, the positive-sense single-stranded genome of ANRSV isolate XC1 was completely determined. The genome annotation revealed the existence of two cysteine proteinases in tandem (HC-Pro1 and HC-Pro2) in the genomic 5' terminus of ANRSV. Sequence comparison and phylogenetic analysis suggested the taxonomic classification of ANRSV into the recently proposed genus Arepavirus in the family Potyviridae. Given the close relationship of ANRSV with another newly reported arepavirus (areca palm necrotic spindle-spot virus), the exact taxonomic status of ANRSV needs to be further investigated. In this study, a reverse transcription polymerase chain reaction assay for ANRSV-specific detection was developed and a close association between ANRSV and ANRSD was found.

A viral suppressor of RNA silencing inhibits ARGONAUTE 1 function by precluding target RNA binding to pre-assembled RISC.

In most eukaryotes, RNA silencing is an adaptive immune system regulating key biological processes including antiviral defense. To evade this response, viruses of plants, worms and insects have evolved viral suppressors of RNA silencing proteins (VSRs). Various VSRs, such as P1 from Sweet potato mild mottle virus (SPMMV), inhibit the activity of RNA-induced silencing complexes (RISCs) including an ARGONAUTE (AGO) protein loaded with a small RNA. However, the specific mechanisms explaining this class of inhibition are unknown. Here, we show that SPMMV P1 interacts with AGO1 and AGO2 from Arabidopsis thaliana, but solely interferes with AGO1 function. Moreover, a mutational analysis of a newly identified zinc finger domain in P1 revealed that this domain could represent an effector domain as it is required for P1 suppressor activity but not for AGO1 binding. Finally, a comparative analysis of the target RNA binding capacity of AGO1 in the presence of wild-type or suppressor-defective P1 forms revealed that P1 blocks target RNA binding to AGO1. Our results describe the negative regulation of RISC, the small RNA containing molecular machine.

Metagenomic analysis of viruses associated with maize lethal necrosis in Kenya.

BACKGROUND: Maize lethal necrosis is caused by a synergistic co-infection of Maize chlorotic mottle virus (MCMV) and a specific member of the Potyviridae, such as Sugarcane mosaic virus (SCMV), Wheat streak mosaic virus (WSMV) or Johnson grass mosaic virus (JGMV). Typical maize lethal necrosis symptoms include severe yellowing and leaf drying from the edges. In Kenya, we detected plants showing typical and atypical symptoms. Both groups of plants often tested negative for SCMV by ELISA. METHODS: We used next-generation sequencing to identify viruses associated to maize lethal necrosis in Kenya through a metagenomics analysis. Symptomatic and asymptomatic leaf samples were collected from maize and sorghum representing sixteen counties. RESULTS: Complete and partial genomes were assembled for MCMV, SCMV, Maize streak virus (MSV) and Maize yellow dwarf virus-RMV (MYDV-RMV). These four viruses (MCMV, SCMV, MSV and MYDV-RMV) were found together in 30 of 68 samples. A geographic analysis showed that these viruses are widely distributed in Kenya. Phylogenetic analyses of nucleotide sequences showed that MCMV, MYDV-RMV and MSV are similar to isolates from East Africa and other parts of the world. Single nucleotide polymorphism, nucleotide and polyprotein sequence alignments identified three genetically distinct groups of SCMV in Kenya. Variation mapped to sequences at the border of NIb and the coat protein. Partial genome sequences were obtained for other four potyviruses and one polerovirus. CONCLUSION: Our results uncover the complexity of the maize lethal necrosis epidemic in Kenya. MCMV, SCMV, MSV and MYDV-RMV are widely distributed and infect both maize and sorghum. SCMV population in Kenya is diverse and consists of numerous strains that are genetically different to isolates from other parts of the world. Several potyviruses, and possibly poleroviruses, are also involved.

The Coat Protein and NIa Protease of Two Potyviridae Family Members Independently Confer Superinfection Exclusion.

Superinfection exclusion (SIE) is an antagonistic virus-virus interaction whereby initial infection by one virus prevents subsequent infection by closely related viruses. Although SIE has been described in diverse viruses infecting plants, humans, and animals, its mechanisms, including involvement of specific viral determinants, are just beginning to be elucidated. In this study, SIE determinants encoded by two economically important wheat viruses, Wheat streak mosaic virus (WSMV; genus Tritimovirus, family Potyviridae) and Triticum mosaic virus (TriMV; genus Poacevirus, family Potyviridae), were identified in gain-of-function experiments that used heterologous viruses to express individual virus-encoded proteins in wheat. Wheat plants infected with TriMV expressing WSMV P1, HC-Pro, P3, 6K1, CI, 6K2, NIa-VPg, or NIb cistrons permitted efficient superinfection by WSMV expressing green fluorescent protein (WSMV-GFP). In contrast, wheat infected with TriMV expressing WSMV NIa-Pro or coat protein (CP) substantially excluded superinfection by WSMV-GFP, suggesting that both of these cistrons are SIE effectors encoded by WSMV. Importantly, SIE is due to functional WSMV NIa-Pro or CP rather than their encoding RNAs, as altering the coded protein products by minimally changing RNA sequences led to abolishment of SIE. Deletion mutagenesis further revealed that elicitation of SIE by NIa-Pro requires the entire protein while CP requires only a 200-amino-acid (aa) middle fragment (aa 101 to 300) of the 349 aa. Strikingly, reciprocal experiments with WSMV-mediated expression of TriMV proteins showed that TriMV CP, and TriMV NIa-Pro to a lesser extent, likewise excluded superinfection by TriMV-GFP. Collectively, these data demonstrate that WSMV- and TriMV-encoded CP and NIa-Pro proteins are effectors of SIE and that these two proteins trigger SIE independently of each other. IMPORTANCE: Superinfection exclusion (SIE) is an antagonistic virus-virus interaction that prevents secondary invasions by identical or closely related viruses in the same host cells. Although known to occur in diverse viruses, SIE remains an enigma in terms of key molecular determinants and action mechanisms. In this study, we found that Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) encode two independently functioning cistrons that serve as effectors of SIE at the protein but not the RNA level. The coat protein and NIa-Pro encoded by these two viruses, when expressed from a heterologous virus, exerted SIE to the cognate viruses. The identification of virus-encoded effectors of SIE and their transgenic expression could potentially facilitate the development of virus-resistant crop plants. Additionally, functional conservation of SIE in diverse virus groups suggests that a better understanding of the underlying mechanisms of SIE could facilitate the development of novel antiviral therapies against viral diseases.

Formation of Potato Virus A-Induced RNA Granules and Viral Translation Are Interrelated Processes Required for Optimal Virus Accumulation.

RNA granules are cellular structures, which play an important role in mRNA translation, storage, and degradation. Animal (+)RNA viruses often co-opt RNA granule proteins for viral reproduction. However, the role of RNA granules in plant viral infections is poorly understood. Here we use Potato virus A (PVA) as a model potyvirus and demonstrate that the helper component-proteinase (HCpro), the potyviral suppressor of RNA silencing, induces the formation of RNA granules. We used confocal microscopy to demonstrate the presence of host RNA binding proteins including acidic ribosomal protein P0, argonaute 1 (AGO1), oligouridylate-binding protein 1 (UBP1), varicose (VCS) and eukaryotic initiation factor iso4E (eIF(iso)4E) in these potyvirus-induced RNA granules. We show that the number of potyviral RNA granules is down-regulated by the genome-linked viral protein (VPg). We demonstrated previously that VPg is a virus-specific translational regulator that co-operates with potyviral RNA granule components P0 and eIF(iso)4E in PVA translation. In this study we show that HCpro and varicose, components of potyviral RNA granules, stimulate VPg-promoted translation of the PVA, whereas UBP1 inhibits this process. Hence, we propose that PVA translation operates via a pathway that is interrelated with potyviral RNA granules in PVA infection. The importance of these granules is evident from the strong reduction in viral RNA and coat protein amounts that follows knock down of potyviral RNA granule components. HCpro suppresses antiviral RNA silencing during infection, and our results allow us to propose that this is also the functional context of the potyviral RNA granules we describe in this study.

PROTEIN DISULFIDE ISOMERASE LIKE 5-1 is a susceptibility factor to plant viruses.

Protein disulfide isomerases (PDIs) catalyze the correct folding of proteins and prevent the aggregation of unfolded or partially folded precursors. Whereas suppression of members of the PDI gene family can delay replication of several human and animal viruses (e.g., HIV), their role in interactions with plant viruses is largely unknown. Here, using a positional cloning strategy we identified variants of PROTEIN DISULFIDE ISOMERASE LIKE 5-1 (HvPDIL5-1) as the cause of naturally occurring resistance to multiple strains of Bymoviruses. The role of wild-type HvPDIL5-1 in conferring susceptibility was confirmed by targeting induced local lesions in genomes for induced mutant alleles, transgene-induced complementation, and allelism tests using different natural resistance alleles. The geographical distribution of natural genetic variants of HvPDIL5-1 revealed the origin of resistance conferring alleles in domesticated barley in Eastern Asia. Higher sequence diversity was correlated with areas with increased pathogen diversity suggesting adaptive selection for bymovirus resistance. HvPDIL5-1 homologs are highly conserved across species of the plant and animal kingdoms implying that orthologs of HvPDIL5-1 or other closely related members of the PDI gene family may be potential susceptibility factors to viruses in other eukaryotic species.

In Vitro Transcripts of Wild-Type and Fluorescent Protein-Tagged Triticum mosaic virus (Family Potyviridae) are Biologically Active in Wheat.

Triticum mosaic virus (TriMV) (genus Poacevirus, family Potyviridae) is a recently described eriophyid mite-transmitted wheat virus. In vitro RNA transcripts generated from full-length cDNA clones of TriMV proved infectious on wheat. Wheat seedlings inoculated with in vitro transcripts elicited mosaic and mottling symptoms similar to the wild-type virus, and the progeny virus was efficiently transmitted by wheat curl mites, indicating that the cloned virus retained pathogenicity, movement, and wheat curl mite transmission characteristics. A series of TriMV-based expression vectors was constructed by engineering a green fluorescent protein (GFP) or red fluorescent protein (RFP) open reading frame with homologous NIa-Pro cleavage peptides between the P1 and HC-Pro cistrons. We found that GFP-tagged TriMV with seven or nine amino acid cleavage peptides efficiently processed GFP from HC-Pro. TriMV-GFP vectors were stable in wheat for more than 120 days and for six serial passages at 14-day intervals by mechanical inoculation and were transmitted by wheat curl mites similarly to the wild-type virus. Fluorescent protein-tagged TriMV was observed in wheat leaves, stems, and crowns. The availability of fluorescent protein-tagged TriMV will facilitate the examination of virus movement and distribution in cereal hosts and the mechanisms of cross protection and synergistic interactions between TriMV and Wheat streak mosaic virus.

Selection pressure and founder effects constrain genetic variation in differentiated populations of soilborne bymovirus Wheat yellow mosaic virus (Potyviridae) in China.

To study the population genetic structure and forces driving the evolution of Wheat yellow mosaic virus (WYMV), the nucleotide sequences encoding the coat protein (CP) (297 sequences) or the genome-linked virion protein (VPg) (87 sequences) were determined from wheat plants growing at 11 different locations distributed in five provinces in China. There were close phylogenetic relationships between all sequences but clustering on the phylogenetic trees was congruent with their provenance, suggesting an origin-dependent population genetic structure. There were low levels of genetic diversity, ranging from 0.00035 ± 0.00019 to 0.01536 ± 0.00043 (CP), and 0.00086 ± 0.00039 to 0.00573 ± 0.00111 (VPg), indicating genetic stability or recent emergence of WYMV in China. The results may suggest that founder effects play a role in shaping the genetic structure of WYMV. Between-population diversity was consistently higher than within-population diversity, suggesting limited gene flow between subpopulations (average FST 0.6241 for the CP and 0.7981 for the VPg). Consistent amino acid substitutions correlated with the provenance of the sequences were observed at nine positions in the CP (but none in the VPg), indicating an advanced stage in population structuring. Strong negative (purifying) selection was implicated on both the CP and VPg but positive selection on a few codons in the CP, indicating an ongoing molecular adaptation.

The N-terminal region of wheat streak mosaic virus coat protein is a host- and strain-specific long-distance transport factor.

Understanding the genetics underlying host range differences among plant virus strains can provide valuable insights into viral gene functions and virus-host interactions. In this study, we examined viral determinants and mechanisms of differential infection of Zea mays inbred line SDp2 by Wheat streak mosaic virus (WSMV) isolates. WSMV isolates Sidney 81 (WSMV-S81) and Type (WSMV-T) share 98.7% polyprotein sequence identity but differentially infect SDp2: WSMV-S81 induces a systemic infection, but WSMV-T does not. Coinoculation and sequential inoculation of SDp2 with WSMV-T and/or WSMV-S81 did not affect systemic infection by WSMV-S81, suggesting that WSMV-T does not induce a restrictive defense response but that virus-encoded proteins may be involved in differential infection of SDp2. The viral determinant responsible for strain-specific host range was mapped to the N terminus of coat protein (CP) by systematic exchanges of WSMV-S81 sequences with those of WSMV-T and by reciprocal exchanges of CP or CP codons 1 to 74. Green fluorescent protein (GFP)-tagged WSMV-S81 with CP or CP residues 1 to 74 from WSMV-T produced similar numbers of infection foci and genomic RNAs and formed virions in inoculated leaves as those produced with WSMV-S81, indicating that failure to infect SDp2 systemically is not due to defects in replication, cell-to-cell movement, or virion assembly. However, these GFP-tagged hybrids showed profound defects in long-distance transport of virus through the phloem. Furthermore, we found that four of the five differing amino acids in the N terminus of CP between the WSMV-S81 and WSMV-T isolates were collectively involved in systemic infection of SDp2. Taken together, these results demonstrate that the N-terminal region of tritimoviral CP functions in host- and strain-specific long-distance movement.

Early detection of plant virus infection using multispectral imaging and spatial-spectral machine learning.

Cassava brown streak disease (CBSD) is an emerging viral disease that can greatly reduce cassava productivity, while causing only mild aerial symptoms that develop late in infection. Early detection of CBSD enables better crop management and intervention. Current techniques require laboratory equipment and are labour intensive and often inaccurate. We have developed a handheld active multispectral imaging (A-MSI) device combined with machine learning for early detection of CBSD in real-time. The principal benefits of A-MSI over passive MSI and conventional camera systems are improved spectral signal-to-noise ratio and temporal repeatability. Information fusion techniques further combine spectral and spatial information to reliably identify features that distinguish healthy cassava from plants with CBSD as early as 28 days post inoculation on a susceptible and a tolerant cultivar. Application of the device has the potential to increase farmers' access to healthy planting materials and reduce losses due to CBSD in Africa. It can also be adapted for sensing other biotic and abiotic stresses in real-world situations where plants are exposed to multiple pest, pathogen and environmental stresses.

An exceptionally high nucleotide and haplotype diversity and a signature of positive selection for the eIF4E resistance gene in barley are revealed by allele mining and phylogenetic analyses of natural populations.

In barley, the eukaryotic translation initiation factor 4E (eIF4E) gene situated on chromosome 3H is recognized as an important source of resistance to the bymoviruses Barley yellow mosaic virus and Barley mild mosaic virus. In modern barley cultivars, two recessive eIF4E alleles, rym4 and rym5, confer different isolate-specific resistances. In this study, the sequence of eIF4E was analysed in 1090 barley landraces and noncurrent cultivars originating from 84 countries. An exceptionally high nucleotide diversity was evident in the coding sequence of eIF4E but not in either the adjacent MCT-1 gene or the sequence-related eIF(iso)4E gene situated on chromosome 1H. Surprisingly, all nucleotide polymorphisms detected in the coding sequence of eIF4E resulted in amino acid changes. A total of 47 eIF4E haplotypes were identified, and phylogenetic analysis using maximum likelihood provided evidence of strong positive selection acting on this barley gene. The majority of eIF4E haplotypes were found to be specific to distinct geographic regions. Furthermore, the eI4FE haplotype diversity (uh) was found to be considerably higher in East Asia, whereas SNP genotyping identified a comparatively low degree of genome-wide genetic diversity in 16 of 17 tested accessions (each carrying a different eIF4E haplotype) from this same region. In addition, selection statistic calculations using coalescent simulations showed evidence of non-neutral variation for eIF4E in several geographic regions, including East Asia, the region with a long history of the bymovirus-induced yellow mosaic disease. Together these findings suggest that eIF4E may play a role in barley adaptation to local habitats.

Generation of transgenic watermelon resistant to Zucchini yellow mosaic virus and Papaya ringspot virus type W.

Zucchini yellow mosaic virus (ZYMV) and Papaya ringspot virus type W (PRSV W) are major limiting factors for production of watermelon worldwide. For the effective control of these two viruses by transgenic resistance, an untranslatable chimeric construct containing truncated ZYMV coat protein (CP) and PRSV W CP genes was transferred to commercial watermelon cultivars by Agrobacterium-mediated transformation. Using our protocol, a total of 27 putative transgenic lines were obtained from three cultivars of 'Feeling' (23 lines), 'China baby' (3 lines), and 'Quality' (1 line). PCR and Southern blot analyses confirmed that the chimeric construct was incorporated into the genomic DNA of the transformants. Greenhouse evaluation of the selected ten transgenic lines of 'Feeling' cultivar revealed that two immune lines conferred complete resistance to ZYMV and PRSV W, from which virus accumulation were not detected by Western blotting 4 weeks after inoculation. The transgenic transcript was not detected, but small interfering RNA (siRNA) was readily detected from the two immune lines and T(1) progeny of line ZW 10 before inoculation, indicating that RNA-mediated post-transcriptional gene silencing (PTGS) is the underlying mechanism for the double-virus resistance. The segregation ratio of T(1) progeny of the immune line ZW10 indicated that the single inserted transgene is nuclearly inherited and associated with the phenotype of double-virus resistance as a dominant trait. The transgenic lines derived from the commercial watermelon cultivars have great potential for control of the two important viruses and can be implemented directly without further breeding.