Dynamics of the rice yellow mottle disease in western Burkina Faso: Epidemic monitoring, spatio-temporal variation of viral diversity, and pathogenicity in a disease hotspot.

The rice yellow mottle virus (RYMV) is a model in plant virus molecular epidemiology, with the reconstruction of historical introduction routes at the scale of the African continent. However, information on patterns of viral prevalence and viral diversity over multiple years at a local scale remains scarce, in spite of potential implications for crop protection. Here, we describe a 5-year (2015-9) monitoring of RYMV prevalence in six sites from western Burkina Faso (geographic areas of Bama, Banzon, and Karfiguela). It confirmed one irrigated site as a disease hotspot and also found one rainfed lowland (RL) site with occasional high prevalence levels. Within the studied fields, a pattern of disease aggregation was evidenced at a 5-m distance, as expected for a mechanically transmitted virus. Next, we monitored RYMV genetic diversity in the irrigated disease hotspot site, revealing a high viral diversity, with the current coexistence of various distinct genetic groups at the site scale (ca. 520 ha) and also within various specific fields (25 m side). One genetic lineage, named S1bzn, is the most recently emerged group and increased in frequency over the studied period (from 20 per cent or less in 2015-6 to more than 65 per cent in 2019). Its genome results from a recombination between two other lineages (S1wa and S1ca). Finally, experimental work revealed that three rice varieties commonly cultivated in Burkina Faso were not different in terms of resistance level, and we also found no significant effect of RYMV genetic groups on symptom expression and viral load. We found, however, that infection outcome depended on the specific RYMV isolate, with two isolates from the lineage S1bzn accumulating at the highest level at early infections. Overall, this study documents a case of high viral prevalence, high viral diversity, and co-occurrence of divergent genetic lineages at a small geographic scale. A recently emerged lineage, which comprises viral isolates inducing severe symptoms and high accumulation under controlled conditions, could be recently rising through natural selection. Following up the monitoring of RYMV diversity is required to confirm this trend and further understand the factors driving the local maintenance of viral diversity.

Spatiotemporal Survey of Multiple Rice Diseases in Irrigated Areas Compared to Rainfed Lowlands in the Western Burkina Faso.

Multiple constraints affect rice yields in West Africa. Among these constraints are viral, bacterial, and fungal pathogens. We aimed to describe the spatiotemporal patterns of occurrence and incidence of multiple rice diseases in farmers' fields in contrasting rice growing systems in the western Burkina Faso. For this purpose, we selected a set of three pairs of sites, each comprising an irrigated area and a neighboring rainfed lowland, and studied them over four consecutive years. We first performed interviews with the rice farmers to better characterize the management practices at the different sites. This study revealed that the transplanting of rice and the possibility of growing rice twice a year are restricted to irrigated areas, while other practices, such as the use of registered rice cultivars, fertilization, and pesticides, are not specific but differ between the two rice growing systems. Then, we performed symptom observations at these study sites to monitor the following four diseases: yellow mottle disease, Bacterial Leaf Streak (BLS), rice leaf blast, and brown spot. The infection rates were found to be higher in irrigated areas than in rainfed lowlands, both when analyzing all observed symptoms together (any of the four diseases) and when specifically considering each of the two diseases: BLS and rice leaf blast. Brown spot was particularly prevalent in all six study sites, while yellow mottle disease was particularly structured geographically. Various diseases were frequently found together in the same field (co-occurrence) or even on the same plant (coinfection), especially in irrigated areas.

Genetic Diversity of Rice stripe necrosis virus and New Insights into Evolution of the Genus Benyvirus.

The rice stripe necrosis virus (RSNV) has been reported to infect rice in several countries in Africa and South America, but limited genomic data are currently publicly available. Here, eleven RSNV genomes were entirely sequenced, including the first corpus of RSNV genomes of African isolates. The genetic variability was differently distributed along the two genomic segments. The segment RNA1, within which clusters of polymorphisms were identified, showed a higher nucleotidic variability than did the beet necrotic yellow vein virus (BNYVV) RNA1 segment. The diversity patterns of both viruses were similar in the RNA2 segment, except for an in-frame insertion of 243 nucleotides located in the RSNV tgbp1 gene. Recombination events were detected into RNA1 and RNA2 segments, in particular in the two most divergent RSNV isolates from Colombia and Sierra Leone. In contrast to BNYVV, the RSNV molecular diversity had a geographical structure with two main RSNV lineages distributed in America and in Africa. Our data on the genetic diversity of RSNV revealed unexpected differences with BNYVV suggesting a complex evolutionary history of the genus Benyvirus.

First report of Sugarcane Streak Mosaic Virus (SCSMV) infecting sugarcane in Côte d'Ivoire.

Sugarcane streak mosaic virus (SCSMV; Poaceavirus; Potiviridae) is the causal agent of streak mosaic disease of sugarcane (Saccharum interspecific hybrids), a major industrial crop that is widely cultivated in tropical and subtropical regions for sugar and ethanol production. was first reported by Hall et al. (1998) from quarantined germplasm material exhibiting mosaic symptoms imported from Pakistan into the USA. Subsequently, the virus was also reported to occur in most of the Asian countries like Bangladesh, India, Indonesia, Iran, Sri Lanka, Thailand, Vietnam and China (Chatenet et al. 2005; Hema et al. 2008, Kasemsin et al. 2016, Putra et al. 2014, Xu et al. 2010, Moradi et al. 2015; Moradi et al. 2018, Zhang et al. 2018). Until now, there is no report of SCSMV outside the Asian continent. From February to October 2018, sugarcane plants exhibiting symptoms such as irregular yellow and green mosaic, interveinal chlorotic specks, and streaks were observed in Bafing (Borotou-Koro), Marahoué (Zuénoula) and Tchologo (Ferkéssédougou) regions of Côte d'Ivoire (Fig. 1a). Varieties under large-scale commercial cultivation such as R570, R579, SP711406, Co997, Co449, M1176/77, M2593/92, M2580/95, and M1400/86 were all symptomatic. A total of 94 sugarcane leaf samples were collected from these regions and, among those, 82 showed disease symptoms and 12 were symptomless. Samples were first tested for the presence of sugarcane mosaic virus (SCMV), which causes mosaic a disease that is already present in Africa. Serological tests with infected sap using a Double Antibody Sandwich (DAS)- Enzyme Linked Immuno Sorbent Assay (ELISA) kit (DSMZ, RT-0166, Braunschweig, Germany) were negative for SCMV and no amplification product was obtained by RT-PCR using primers specific to the coat protein (CP) gene of SCMV (Putra et al. 2003). The 82 symptomatic leaves tested positive by DAS-ELISA with SCSMV antiserum (polyclonal antibodies were graciously provided by Prof. Hema M. of the Sri Venkateswara University, Tirupati, AP, India), whereas the 12 symptomless samples tested negative. To confirm these results, virus free greenhouse-grown sugarcane varieties Co997 and M1176/77, were mechanically inoculated with 10 sap extracts from 10 SCSMV-infected sugarcane leaf samples. Sap was also extracted from DAS-ELISA negative sugarcane leaves and used as negative control. For sap preparation, leaves were homogenized with a mortar in 2 mL of phosphate buffer 0.01 M pH 7.2 (ratio 1:10). Fifteen 4-week-old plants per variety were inoculated separately with each sap. All inoculated plants exhibited streak mosaic symptoms 13 days post-inoculation (fig. 1b), and the presence of SCSMV in the inoculated plants was confirmed by DAS-ELISA. Total RNA was extracted from four symptomatic leaf samples, one symptomless and one DAS-ELISA positive sample using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized using M-MLV Reverse Transcriptase (Promega, Cat.No.M1705, Madison, WI, USA) following the manufacturer's instructions. A 690-nucleotide fragment of the CP gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) with primers SCSMV-F690 and SCSMV-R690 (Viswanathan et al. 2008). All primers were synthesized by Eurogentec (Seraing, Belgium). Aliquots of RT-PCR products (5 µl) were analyzed by electrophoresis on 1.2 % (w/v) agarose gel, stained with ethidium bromide and visualized on a UV transilluminator (Fig. 2). An amplification product of the expected size was obtained for all five symptomatic or DAS ELISA positive but not for the symptomless sample. Two RT-PCR products were sequenced and deposited in GenBank under accession Nos. LR594547 and LR594582. These partial CP gene sequences shared highest nucleotide identity with two isolates of SCSMV from India in GenBank: 91% with JN315855 and 90% with EF655859, thus confirming that SCSMV was occurring in sugarcane in Côte d'Ivoire. To our knowledge, this is the first report of natural infection of sugarcane by SCSMV in Africa. Streak mosaic is a serious threat to the entire sugar industry in West Africa and needs further investigations as it may affect sugarcane yields and impact local economies. Our findings further illustrate the need to develop virus-free germplasm for local, national, and international distribution of sugarcane.

Nicotiana benthamiana is a suitable transient system for high-level expression of an active inhibitor of cotton boll weevil α-amylase.

BACKGROUND: Insect resistance in crops represents a main challenge for agriculture. Transgenic approaches based on proteins displaying insect resistance properties are widely used as efficient breeding strategies. To extend the spectrum of targeted pathogens and overtake the development of resistance, molecular evolution strategies have been used on genes encoding these proteins to generate thousands of variants with new or improved functions. The cotton boll weevil (Anthonomus grandis) is one of the major pests of cotton in the Americas. An α-amylase inhibitor (α-AIC3) variant previously developed via molecular evolution strategy showed inhibitory activity against A. grandis α-amylase (AGA). RESULTS: We produced in a few days considerable amounts of α-AIC3 using an optimised transient heterologous expression system in Nicotiana benthamiana. This high α-AIC3 accumulation allowed its structural and functional characterizations. We demonstrated via MALDI-TOF MS/MS technique that the protein was processed as expected. It could inhibit up to 100% of AGA biological activity whereas it did not act on α-amylase of two non-pathogenic insects. These data confirmed that N. benthamiana is a suitable and simple system for high-level production of biologically active α-AIC3. Based on other benefits such as economic, health and environmental that need to be considerate, our data suggested that α-AIC3 could be a very promising candidate for the production of transgenic crops resistant to cotton boll weevil without lethal effect on at least two non-pathogenic insects. CONCLUSIONS: We propose this expression system can be complementary to molecular evolution strategies to identify the most promising variants before starting long-lasting stable transgenic programs.

Virus-based pharmaceutical production in plants: an opportunity to reduce health problems in Africa.

BACKGROUND: Developing African countries face health problems that they struggle to solve. The major causes of this situation are high therapeutic and logistical costs. Plant-made therapeutics are easy to produce due to the lack of the safety considerations associated with traditional fermenter-based expression platforms, such as mammalian cells. Plant biosystems are easy to scale up and inexpensive, and they do not require refrigeration or a sophisticated medical infrastructure. These advantages provide an opportunity for plant-made pharmaceuticals to counteract diseases for which medicines were previously inaccessible to people in countries with few resources. MAIN BODY: The techniques needed for plant-based therapeutic production are currently available. Viral expression vectors based on plant viruses have greatly enhanced plant-made therapeutic production and have been exploited to produce a variety of proteins of industrial, pharmaceutical and agribusiness interest. Some neglected tropical diseases occurring exclusively in the developing world have found solutions through plant bioreactor technology. Plant viral expression vectors have been reported in the production of therapeutics against these diseases occurring exclusively in the third world, and some virus-derived antigens produced in plants exhibit appropriate antigenicity and immunogenicity. However, all advances in the use of plants as bioreactors have been made by companies in Europe and America. The developing world is still far from acquiring this technology, although plant viral expression vectors may provide crucial help to overcome neglected diseases. CONCLUSION: Today, interest in these tools is rising, and viral amplicons made in and for Africa are in progress. This review describes the biotechnological advances in the field of plant bioreactors, highlights factors restricting access to this technology by those who need it most and proposes a solution to overcome these limitations.

Optimized transitory ectopic expression of promastigote surface antigen protein in Nicotiana benthamiana, a potential anti-leishmaniasis vaccine candidate.

In recent years, plants have been shown to be an efficient alternative expression system for high-value pharmaceuticals such as vaccines. However, constitutive expression of recombinant protein remains uncertain on their level of production and biological activity. To overcome these problems, transitory expression systems have been developed. Here, a series of experiments were performed to determine the most effective conditions to enhance vaccine antigen transient accumulation in Nicotiana benthamiana leaves using the promastigote surface antigen (PSA) from the parasitic protozoan Leishmania infantum. This protein has been previously identified as the major antigen of a licensed canine anti-leishmaniasis vaccine. The classical prokaryote Escherichia coli biosystem failed in accumulating PSA. Consequently, the standard plant system based on N. benthamiana has been optimized for the production of putatively active PSA. First, the RNA silencing defense mechanism set up by the plant against PSA ectopic expression was abolished by using three viral suppressors acting at different steps of the RNA silencing pathway. Then, we demonstrated that the signal peptide at the N-terminal side of the PSA is required for its accumulation. The PSA ER signaling and retention with the PSA signal peptide and the KDEL motif, respectively were optimized to significantly increase its accumulation. Finally, we demonstrate that the production of recombinant PSA in N. benthamiana leaves allows the conservation of its immunogenic property. These approaches demonstrate that based on these optimizations, plant based systems can be used to effectively produce the biological active PSA protein.

Virus-Bacteria Rice Co-Infection in Africa: Field Estimation, Reciprocal Effects, Molecular Mechanisms, and Evolutionary Implications.

Simultaneous infection of a single plant by various pathogen species is increasingly recognized as an important modulator of host resistance and a driver of pathogen evolution. Because plants in agro-ecosystems are the target of a multitude of pathogenic microbes, co-infection could be frequent, and consequently important to consider. This is particularly true for rapidly intensifying crops, such as rice in Africa. This study investigated potential interactions between pathogens causing two of the major rice diseases in Africa: the Rice yellow mottle virus (RYMV) and the bacterium Xanthomonas oryzae pathovar oryzicola (Xoc) in order to: 1/ document virus-bacteria co-infection in rice in the field, 2/ explore experimentally their consequences in terms of symptom development and pathogen multiplication, 3/ test the hypothesis of underlying molecular mechanisms of interactions and 4/ explore potential evolutionary consequences. Field surveys in Burkina Faso revealed that a significant proportion of rice fields were simultaneously affected by the two diseases. Co-infection leads to an increase in bacterial specific symptoms, while a decrease in viral load is observed compared to the mono-infected mock. The lack of effect found when using a bacterial mutant for an effector specifically inducing expression of a small RNA regulatory protein, HEN1, as well as a viral genotype-specific effect, both suggest a role for gene silencing mechanisms mediating the within-plant interaction between RYMV and Xoc. Potential implications for pathogen evolution could not be inferred because genotype-specific effects were found only for pathogens originating from different countries, and consequently not meeting in the agrosystem. We argue that pathogen-pathogen-host interactions certainly deserve more attention, both from a theoretical and applied point of view.

Sites under positive selection modulate the RNA silencing suppressor activity of rice yellow mottle virus movement protein P1.

RNA silencing is a eukaryotic mechanism for RNA-based gene regulation that plays an essential role in diverse biological processes, such as defence against viral infections. The P1 of rice yellow mottle virus (RYMV) is a movement protein and displays RNA silencing suppression activity with variable efficiency, depending on the origin of the isolates. In this study, the positive selection pressure acting on the P1 protein gene was assessed. A site-by-site analysis of the dN/dS ratio was performed and 18 positively selected sites were identified. Four of these were mutated, and the ability to suppress RNA silencing was evaluated for the resulting mutants in a transient expression assay. All mutations affected quantitatively RNA silencing suppression, one caused a significant decrease in the activity and three significantly increased it. This work demonstrates, for what is to the best of our knowledge the first time, that the RYMV gene encoding the P1 RNA silencing suppressor is under adaptive evolution.

[Plant viruses RNA silencing suppressors: characterization and mechanism of action]. / Les suppresseurs du RNA silencing des phytovirus: caractérisation et mode d'action.

RNA silencing or Post-transcriptional gene silencing (PTGS) in plants is a fundamental defence mechanism against viruses, transgenes and transposons. Most viruses, if not all, are able to overcome RNA-silencing through the production of so-called "silencing suppressors" with counterdefence ability". This strategy is well known for plant and animal viruses. Silencing suppressor proteins block the host RNA silencing by targeting different steps of the silencing pathway. In this review, we will focus on the major silencing suppressor proteins encoded by plant viruses and on the methods used to identify and characterize the molecular bases of silencing suppression.

Biological and molecular characterization of a putative new sobemovirus infecting Imperata cylindrica and maize in Africa.

A new virus was isolated from both the grass Imperata cylindrica and maize plants that had yellow mottle symptoms in Burkina Faso, West Africa. The virus has isometric particles ca. 32 nm in diameter. The experimental host range was restricted to Rottboellia exaltata. Virions were isolated from leaves of systemically infected maize plants. Koch's postulates were completed by mechanically inoculating uninfected Imperata or maize with either purified virus or sap from infected Imperata plants. Virion preparations were used to produce a specific polyclonal antiserum, and an enzyme-linked immunosorbent assay test was set up. The full genome of the virus was sequenced, and it comprised 4,547 nucleotides. Phylogenetic studies indicated that the virus is closely related to rice yellow mottle virus, a sobemovirus that infects monocotyledons in Africa, and is more distantly related to cocksfoot mottle virus, another sobemovirus that infects monocotyledons. Although the virus can infect R. exaltata experimentally, it differs from Rottboellia yellow mottle virus, a member of a tentative species of the genus Sobemovirus that also infects monocotyledons in Africa. Particle morphology, serological properties, genomic organization, and phylogenetic analysis are all consistent with assignment of the new virus to the genus Sobemovirus. The name Imperata yellow mottle virus is proposed.

Diversification of rice yellow mottle virus and related viruses spans the history of agriculture from the neolithic to the present.

The mechanisms of evolution of plant viruses are being unraveled, yet the timescale of their evolution remains an enigma. To address this critical issue, the divergence time of plant viruses at the intra- and inter-specific levels was assessed. The time of the most recent common ancestor (TMRCA) of Rice yellow mottle virus (RYMV; genus Sobemovirus) was calculated by a Bayesian coalescent analysis of the coat protein sequences of 253 isolates collected between 1966 and 2006 from all over Africa. It is inferred that RYMV diversified approximately 200 years ago in Africa, i.e., centuries after rice was domesticated or introduced, and decades before epidemics were reported. The divergence time of sobemoviruses and viruses of related genera was subsequently assessed using the age of RYMV under a relaxed molecular clock for calibration. The divergence time between sobemoviruses and related viruses was estimated to be approximately 9,000 years, that between sobemoviruses and poleroviruses approximately 5,000 years, and that among sobemoviruses approximately 3,000 years. The TMRCA of closely related pairs of sobemoviruses, poleroviruses, and luteoviruses was approximately 500 years, which is a measure of the time associated with plant virus speciation. It is concluded that the diversification of RYMV and related viruses has spanned the history of agriculture, from the Neolithic age to the present.

Theme and variations in the evolutionary pathways to virulence of an RNA plant virus species.

The diversity of a highly variable RNA plant virus was considered to determine the range of virulence substitutions, the evolutionary pathways to virulence, and whether intraspecific diversity modulates virulence pathways and propensity. In all, 114 isolates representative of the genetic and geographic diversity of Rice yellow mottle virus (RYMV) in Africa were inoculated to several cultivars with eIF(iso)4G-mediated Rymv1-2 resistance. Altogether, 41 virulent variants generated from ten wild isolates were analyzed. Nonconservative amino acid replacements at five positions located within a stretch of 15 codons in the central region of the 79-aa-long protein VPg were associated with virulence. Virulence substitutions were fixed predominantly at codon 48 in most strains, whatever the host genetic background or the experimental conditions. There were one major and two isolate-specific mutational pathways conferring virulence at codon 48. In the prevalent mutational pathway I, arginine (AGA) was successively displaced by glycine (GGA) and glutamic acid (GAA). Substitutions in the other virulence codons were displaced when E48 was fixed. In the isolate-specific mutational pathway II, isoleucine (ATA) emerged and often later coexisted with valine (GTA). In mutational pathway III, arginine, with the specific S2/S3 strain codon usage AGG, was displaced by tryptophane (TGG). Mutational pathway I never arose in the widely spread West African S2/S3 strain because G48 was not infectious in the S2/S3 genetic context. Strain S2/S3 least frequently overcame resistance, whereas two geographically localized variants of the strain S4 had a high propensity to virulence. Codons 49 and 26 of the VPg, under diversifying selection, are candidate positions in modulating the genetic barriers to virulence. The theme and variations in the evolutionary pathways to virulence of RYMV illustrates the extent of parallel evolution within a highly variable RNA plant virus species.