Differential Tropism in Roots and Shoots of Resistant and Susceptible Cassava (Manihot esculenta Crantz) Infected by Cassava Brown Streak Viruses.

Cassava brown streak disease (CBSD) is a destructive disease of cassava in Eastern and Central Africa. Because there was no source of resistance in African varieties to provide complete protection against the viruses causing the disease, we searched in South American germplasm and identified cassava lines that did not become infected with the cassava brown streak viruses. These findings motivated further investigations into the mechanism of virus resistance. We used RNAscope® in situ hybridization to localize cassava brown streak virus in cassava germplasm lines that were highly resistant (DSC 167, immune) or that restricted virus infections to stems and roots only (DSC 260). We show that the resistance in those lines is not a restriction of long-distance movement but due to preventing virus unloading from the phloem into parenchyma cells for replication, thus restricting the virus to the phloem cells only. When DSC 167 and DSC 260 were compared for virus invasion, only a low CBSV signal was found in phloem tissue of DSC 167, indicating that there is no replication in this host, while the presence of intense hybridization signals in the phloem of DSC 260 provided evidence for virus replication in companion cells. In neither of the two lines studied was there evidence of virus replication outside the phloem tissues. Thus, we conclude that in resistant cassava lines, CBSV is confined to the phloem tissues only, in which virus replication can still take place or is arrested.

Complete genome sequence and construction of an infectious full-length cDNA clone of celery latent virus - an unusual member of a putative new genus within the Potyviridae.

Celery latent virus (CeLV) is an incompletely described plant virus known to be sap and seed transmissible and to possess flexuous filamentous particles measuring about 900 nm in length, suggesting it as a possible member of the family Potyviridae. Here, an Italian isolate of CeLV was transmitted by sap to a number of host plants and shown to have a single-stranded and monopartite RNA genome being 11 519 nucleotides (nts) in size and possessing some unusual features. The RNA contains a large open reading frame (ORF) that is flanked by a short 5' untranslated region (UTR) of 13 nt and a 3' UTR consisting of 586 nt that is not polyadenylated. CeLV RNA shares nt sequence identity of only about 40 % with other members of the Potyviridae (potyvirids). The CeLV polyprotein is notable in that it starts with a signal peptide, has a putative P3N-PIPO ORF and shares low aa sequence identity (about 18 %) with other potyvirids. Although potential cleavage sites were not identified for the N-terminal two-thirds of the polyprotein, the latter possesses a number of sequence motifs, the identity and position of which are characteristic of other potyvirids. Attempts at constructing an infectious full-length cDNA clone of CeLV were successful following Rhizobium radiobacter infiltration of Nicotiana benthamiana and Apium graveolens. CeLV appears to have the largest genome of all known potyvirids and some unique genome features that may warrant the creation of a new genus, for which we propose the name 'celavirus'.

Whitefly-transmitted viruses threatening cassava production in Africa.

Emerging plant viruses are one of the greatest problems facing crop production worldwide, and have severe consequences in the developing world where subsistence farming is a major source of food production, and knowledge and resources for management are limited. In Africa, evolution of two viral disease complexes, cassava mosaic begomoviruses (CMBs) (Geminiviridae) and cassava brown streak viruses (CBSVs) (Potyviridae), have resulted in severe pandemics that continue to spread and threaten cassava production. Identification of genetically diverse and rapidly evolving CMBs and CBSVs, extensive genetic variation in the vector, Bemisia tabaci (Hemiptera: Aleyrodidae), and numerous secondary endosymbiont profiles that influence vector phenotypes suggest that complex local and regional vector-virus-plant-environment interactions may be driving the evolution and epidemiology of these viruses.

Maize Lethal Necrosis: An Emerging, Synergistic Viral Disease.

Maize lethal necrosis (MLN) is a disease of maize caused by coinfection of maize with maize chlorotic mottle virus (MCMV) and one of several viruses from the Potyviridae, such as sugarcane mosaic virus, maize dwarf mosaic virus, Johnsongrass mosaic virus or wheat streak mosaic virus. The coinfecting viruses act synergistically to result in frequent plant death or severely reduce or negligible yield. Over the past eight years, MLN has emerged in sub-Saharan East Africa, Southeast Asia, and South America, with large impacts on smallholder farmers. Factors associated with MLN emergence include multiple maize crops per year, the presence of maize thrips ( Frankliniella williamsi), and highly susceptible maize crops. Soil and seed transmission of MCMV may also play significant roles in development and perpetuation of MLN epidemics. Containment and control of MLN will likely require a multipronged approach, and more research is needed to identify and develop the best measures.

A time series transcriptome analysis of cassava (Manihot esculenta Crantz) varieties challenged with Ugandan cassava brown streak virus.

A time-course transcriptome analysis of two cassava varieties that are either resistant or susceptible to cassava brown streak disease (CBSD) was conducted using RNASeq, after graft inoculation with Ugandan cassava brown streak virus (UCBSV). From approximately 1.92 billion short reads, the largest number of differentially expressed genes (DEGs) was obtained in the resistant (Namikonga) variety at 2 days after grafting (dag) (3887 DEGs) and 5 dag (4911 DEGs). At the same time points, several defense response genes (encoding LRR-containing, NBARC-containing, pathogenesis-related, late embryogenesis abundant, selected transcription factors, chaperones, and heat shock proteins) were highly expressed in Namikonga. Also, defense-related GO terms of 'translational elongation', 'translation factor activity', 'ribosomal subunit' and 'phosphorelay signal transduction', were overrepresented in Namikonga at these time points. More reads corresponding to UCBSV sequences were recovered from the susceptible variety (Albert) (733 and 1660 read counts per million (cpm)) at 45 dag and 54 dag compared to Namikonga (10 and 117 cpm respectively). These findings suggest that Namikonga's resistance involves restriction of multiplication of UCBSV within the host. These findings can be used with other sources of evidence to identify candidate genes and biomarkers that would contribute substantially to knowledge-based resistance breeding.

Comparative analysis of virus-derived small RNAs within cassava (Manihot esculenta Crantz) infected with cassava brown streak viruses.

Infection of plant cells by viral pathogens triggers RNA silencing, an innate antiviral defense mechanism. In response to infection, small RNAs (sRNAs) are produced that associate with Argonaute (AGO)-containing silencing complexes which act to inactivate viral genomes by posttranscriptional gene silencing (PTGS). Deep sequencing was used to compare virus-derived small RNAs (vsRNAs) in cassava genotypes NASE 3, TME 204 and 60444 infected with the positive sense single-stranded RNA (+ssRNA) viruses cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), the causal agents of cassava brown streak disease (CBSD). An abundance of 21-24nt vsRNAs was detected and mapped, covering the entire CBSV and UCBSV genomes. The 21nt vsRNAs were most predominant, followed by the 22 nt class with a slight bias toward sense compared to antisense polarity, and a bias for adenine and uracil bases present at the 5'-terminus. Distribution and frequency of vsRNAs differed between cassava genotypes and viral genomes. In susceptible genotypes TME 204 and 60444, CBSV-derived sRNAs were seen in greater abundance than UCBSV-derived sRNAs. NASE 3, known to be resistant to UCBSV, accumulated negligible UCBSV-derived sRNAs but high populations of CBSV-derived sRNAs. Transcript levels of cassava homologues of AGO2, DCL2 and DCL4, which are central to the gene-silencing complex, were found to be differentially regulated in CBSV- and UCBSV-infected plants across genotypes, suggesting these proteins play a role in antiviral defense. Irrespective of genotype or viral pathogen, maximum populations of vsRNAs mapped to the cytoplasmic inclusion, P1 and P3 protein-encoding regions. Our results indicate disparity between CBSV and UCBSV host-virus interaction mechanisms, and provide insight into the role of virus-induced gene silencing as a mechanism of resistance to CBSD.

Heritable, de novo resistance to leaf rust and other novel traits in selfed descendants of wheat responding to inoculation with wheat streak mosaic virus.

Stable resistance to infection with Wheat streak mosaic virus (WSMV) can be evolved de novo in selfing bread wheat lines subjected to cycles of WSMV inoculation and selection of best-performing plants or tillers. To learn whether this phenomenon might be applied to evolve resistance de novo to pathogens unrelated to WSMV, we examined the responses to leaf rust of succeeding generations of the rust- and WSMV-susceptible cultivar 'Lakin' following WSMV inoculation and derived rust-resistant sublines. After three cycles of the iterative protocol five plants, in contrast to all others, expressed resistance to leaf and stripe rust. A subset of descendant sublines of one of these, 'R1', heritably and uniformly expressed the new trait of resistance to leaf rust. Such sublines, into which no genes from a known source of resistance had been introgressed, conferred resistance to progeny of crosses with susceptible parents. The F1 populations produced from crosses between, respectively, susceptible and resistant 'Lakin' sublines 4-3-3 and 4-12-3 were not all uniform in their response to seedling inoculation with race TDBG. In seedling tests against TDBG and MKPS races the F2s from F1 populations that were uniformly resistant had 3∶1 ratios of resistant to susceptible individuals but the F2s from susceptible F1 progenitors were uniformly susceptible. True-breeding lines derived from resistant individuals in F2 populations were resistant to natural stripe and leaf rust inoculum in the field, while the 'Lakin' progenitor was susceptible. The next generation of six of the 'Lakin'-derived lines exhibited moderate to strong de novo resistance to stem rust races TPMK, QFCS and RKQQ in seedling tests while the 'Lakin' progenitor was susceptible. These apparently epigenetic effects in response to virus infection may help researchers fashion a new tool that expands the range of genetic resources already available in adapted germplasm.

Low genetic diversity of Squash vein yellowing virus in wild and cultivated cucurbits in the U.S. suggests a recent introduction.

Squash vein yellowing virus (SqVYV) isolates were collected from cultivated and weedy cucurbits representing major hosts and locations in the U.S. and analyzed to better understand the diversity and population structure. No differences in symptoms were observed in field-collected isolate source plants or subsequently inoculated greenhouse plants, and the complete genome of an SqVYV isolate from a wild cucurbit host (smellmelon, Cucumis melo var. dudaim) was highly similar (99.4% nucleotide identity, 99.3% amino acid identity) to the previously published type isolate from squash. Although analysis of the coat protein (CP) and two serine proteases (P1a and P1b) sequences for 41 isolates showed little diversity across seven years of sampling, it revealed two distinct groups of SqVYV isolates with low intra-group diversity. Our analyses also suggested that recombination had occurred between SqVYV isolates, similar to other ipomoviruses. Selection pressures on the genome regions analyzed were negative indicating purifying selection was occurring. The magnitude of negative selection in SqVYV was consistent with what has been reported for other ipomoviruses, and was greatest for the CP and least for the P1b. The observed genetic diversity was similar to that reported for Cucumber vein yellowing virus but less than that reported for Sweet potato mild mottle virus, Cassava brown streak virus and Ugandan cassava brown streak virus. Collectively, these results indicate that the current U.S. population of SqVYV has undergone a recent genetic bottleneck and was introduced from elsewhere.

Yellowing disease in zucchini squash produced by mixed infections of Cucurbit yellow stunting disorder virus and Cucumber vein yellowing virus.

Zucchini squash is host to Cucurbit yellow stunting disorder virus (CYSDV), a member of the genus Crinivirus, and Cucumber vein yellowing virus (CVYV), a member of the genus Ipomovirus, both transmitted by the whitefly Bemisia tabaci. Field observations suggest the appearance of new symptoms observed on leaves of zucchini squash crops when both viruses were present. When infected during controlled experiments with CYSDV only, zucchini plants showed no obvious symptoms and the virus titer decreased between 15 and 45 days postinoculation (dpi), after which it was no longer detected. CVYV caused inconspicuous symptoms restricted to vein clearing on some of the apical leaves and the virus accumulated progressively between 15 and 60 dpi. Similar accumulations of virus followed single inoculations with the potyvirus Zucchini yellow mosaic virus (ZYMV) and plants showed severe stunting, leaf deformation, and mosaic yellowing. However, in mixed infections with CYSDV and CVYV, intermediate leaves showed chlorotic mottling which evolved later to rolling, brittleness, and complete yellowing of the leaf lamina, with exception of the veins. No consistent alteration of CVYV accumulation was detected but the amounts of CYSDV increased ≈100-fold and remained detectable at 60 dpi. Such synergistic effects on the titer of the crinivirus and symptom expression were not observed when co-infected with ZYMV.

Tempo and mode of plant RNA virus escape from RNA interference-mediated resistance.

A biotechnological application of artificial microRNAs (amiRs) is the generation of plants that are resistant to virus infection. This resistance has proven to be highly effective and sequence specific. However, before these transgenic plants can be deployed in the field, it is important to evaluate the likelihood of the emergence of resistance-breaking mutants. Two issues are of particular interest: (i) whether such mutants can arise in nontransgenic plants that may act as reservoirs and (ii) whether a suboptimal expression level of the transgene, resulting in subinhibitory concentrations of the amiR, would favor the emergence of escape mutants. To address the first issue, we experimentally evolved independent lineages of Turnip mosaic virus (TuMV) (family Potyviridae) in fully susceptible wild-type Arabidopsis thaliana plants and then simulated the spillover of the evolving virus to fully resistant A. thaliana transgenic plants. To address the second issue, the evolution phase took place with transgenic plants that expressed the amiR at subinhibitory concentrations. Our results show that TuMV populations replicating in susceptible hosts accumulated resistance-breaking alleles that resulted in the overcoming of the resistance of fully resistant plants. The rate at which resistance was broken was 7 times higher for TuMV populations that experienced subinhibitory concentrations of the antiviral amiR. A molecular characterization of escape alleles showed that they all contained at least one nucleotide substitution in the target sequence, generally a transition of the G-to-A and C-to-U types, with many instances of convergent molecular evolution. To better understand the viral population dynamics taking place within each host, as well as to evaluate relevant population genetic parameters, we performed in silico simulations of the experiments. Together, our results contribute to the rational management of amiR-based antiviral resistance in plants.

Elimination of two viruses which interact synergistically from sweetpotato by shoot tip culture and cryotherapy.

Sweet potato chlorotic stunt virus (SPCSV; Closteroviridae) and Sweet potato feathery mottle virus (SPFMV; Potyviridae) interact synergistically and cause severe diseases in co-infected sweetpotato plants (Ipomoea batatas). Sweetpotato is propagated vegetatively and virus-free planting materials are pivotal for sustainable production. Using cryotherapy, SPCSV and SPCSV were eliminated from all treated single-virus-infected and co-infected shoot tips irrespective of size (0.5-1.5mm including 2-4 leaf primordia). While shoot tip culture also eliminated SPCSV, elimination of SPFMV failed in 90-93% of the largest shoot tips (1.5mm) using this technique. Virus distribution to different leaf primordia and tissues within leaf primordia in the shoot apex and petioles was not altered by co-infection of the viruses in the fully virus-susceptible sweetpotato genotype used. SPFMV was immunolocalized to all types of tissues and up to the fourth-youngest leaf primordium. In contrast, SPCSV was detected only in the phloem and up to the fifth leaf primordium. Because only cells in the apical dome of the meristem and the two first leaf primordia survived cryotherapy, all data taken together could explain the results of virus elimination. The simple and efficient cryotherapy protocol developed for virus elimination can also be used for preparation of sweetpotato materials for long-term preservation.

[Inheritance analysis and molecular marker selection of genes for wheat spindle streak mosaic disease resistance].

Three wheat spindle streak mosaic viruses (WSSMV) resistant cultivars ('Yining Xiaomai', 'Xu87-633', and 'Xifeng') and one susceptible cultivar ('Zhen9523') were used as parents of 3 crosses in this experiment. WSSMV resistance of the parents, F1, and F2 was evaluated under field condition. Based on the segregation ratios of resistant and susceptible plants in F, and F2 populations, it was deduced that the resistance to WSSMV was dominant and the inheritable factors controlling WSSMV resistance were encoded by the nuclear genome. WSSMV resistances in 'Yining Xiaomai' and 'Xifeng' were controlled by two pairs of alleles, which showed complementary effects. However the resistance in 'Xu-87633' was controlled by a single dominant gene. 266 pairs of SSR primers located on 21 wheat chromosomes were used for polymorphic analysis of the two resistant and the susceptible parents 'Yining Xiaomai' and 'Zhen9523', and 108 of them amplified polymorphic DNA products. By Bulk Segregant Analysis of resistant and susceptible pools, one pair of primer located on chromosome arm 2DS, Xgwm261, were found being linked to WSSMV resistance. The 224 F2 individuals were then amplified with marker Xgwm261. The statistic genetic distance between Xgwm261 and the resistance locus was calculated to be 22.9 cM using the software Mapmaker 3.0.