RNAi-mediated resistance to diverse isolates belonging to two virus species involved in Cassava brown streak disease

Cassava brown streak disease (CBSD) is emerging as one of the most important viral diseases of cassava (Manihot esculenta) and is considered today as the biggest threat to cassava cultivation in East Africa. The disease is caused by isolates of at least two phylogenetically distinct species of single-stranded RNA viruses belonging to the family Potyviridae, genus Ipomovirus. The two species are present predominantly in the coastal lowland [Cassava brown streak virus (CBSV); Tanzania and Mozambique] and highland [Cassava brown streak Uganda virus (CBSUV); Lake Victoria Basin, Uganda, Kenya and Malawi] in East Africa. In this study, we demonstrate that CBSD can be efficiently controlled using RNA interference (RNAi). Three RNAi constructs targeting the highland species were generated, consisting of the full-length (FL; 894 nucleotides), 397-nucleotide N-terminal and 491-nucleotide C-terminal portions of the coat protein (CP) gene of a Ugandan isolate of CBSUV (CBSUV-[UG:Nam:04]), and expressed constitutively in Nicotiana benthamiana. After challenge with CBSUV-[UG:Nam:04], plants homozygous for FL-CP showed the highest resistance, followed by the N-terminal and C-terminal lines with similar resistance. In the case of FL, approximately 85% of the transgenic plant lines produced were completely resistant. Some transgenic lines were also challenged with six distinct isolates representing both species: CBSV and CBSUV. In addition to nearly complete resistance to the homologous virus, two FL plant lines showed 100% resistance and two C-terminal lines expressed 50-100% resistance, whereas the N-terminal lines succumbed to the nonhomologous CBSV isolates. Northern blotting revealed a positive correlation between the level of transgene-specific small interfering RNAs detected in transgenic plants and the level of virus resistance. This is the first demonstration of RNAi-mediated resistance to CBSD and protection across very distant isolates (more than 25% in nucleotide sequence) belonging to two different species: Cassava brown streak virus and Cassava brown streak Uganda virus.     

Cassava brown streak virus Ham1 protein hydrolyses mutagenic nucleotides and is a necrosis determinant

Cassava brown streak disease (CBSD) is a leading cause of cassava losses in East and Central Africa, and is currently having a severe impact on food security. The disease is caused by two viruses within the Potyviridae family: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), which both encode atypical Ham1 proteins with highly conserved inosine triphosphate (ITP) pyrophosphohydrolase (ITPase) domains. ITPase proteins are widely encoded by plant, animal, and archaea. They selectively hydrolyse mutagenic nucleotide triphosphates to prevent their incorporation into nucleic acid and thereby function to reduce mutation rates. It has previously been hypothesized that U/CBSVs encode Ham1 proteins with ITPase activity to reduce viral mutation rates during infection. In this study, we investigate the potential roles of U/CBSV Ham1 proteins. We show that both CBSV and UCBSV Ham1 proteins have ITPase activities through in vitro enzyme assays. Deep-sequencing experiments found no evidence of the U/CBSV Ham1 proteins providing mutagenic protection during infections of Nicotiana hosts. Manipulations of the CBSV_Tanza infectious clone were performed, including a Ham1 deletion, ITPase point mutations, and UCBSV Ham1 chimera. Unlike severely necrotic wild-type CBSV_Tanza infections, infections of Nicotiana benthamiana with the manipulated CBSV infectious clones do not develop necrosis, indicating that that the CBSV Ham1 is a necrosis determinant. We propose that the presence of U/CBSV Ham1 proteins with highly conserved ITPase motifs indicates that they serve highly selectable functions during infections of cassava and may represent a euphorbia host adaptation that could be targeted in antiviral strategies.     

Dose-dependent RNAi-mediated geminivirus resistance in the tropical root crop cassava

Cassava mosaic disease is a major constraint for cassava production in Africa, resulting in significant economic losses. We have engineered transgenic cassava with resistance to African cassava mosaic virus (ACMV), by expressing ACMV AC1-homologous hairpin double-strand RNAs. Transgenic cassava lines with high levels of AC1-homologous small RNAs have ACMV immunity with increasing viral load and different inoculation methods. We report a correlation between the expression of the AC1-homologous small RNAs and the ACMV resistance of the transgenic cassava lines. Characterization of the small RNAs revealed that only some of the hairpin-derived small RNAs fall into currently known small interfering RNA classes in plants. The method is scalable to stacking by targeting multiple virus isolates with additional hairpins. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

QTL associated with resistance to cassava brown streak and cassava mosaic diseases in a bi-parental cross of two Tanzanian farmer varieties,Namikonga and Albert

Key message

QTL consistent across seasons were detected for resistance to cassava brown streak disease induced root necrosis and foliar symptoms. The CMD2 locus was detected in an East African landrace, and comprised two QTL.

Abstract

Cassava production in Africa is compromised by cassava brown streak disease (CBSD) and cassava mosaic disease (CMD). To reduce costs and increase the precision of resistance breeding, a QTL study was conducted to identify molecular markers linked to resistance against these diseases. A bi-parental F₁ mapping population was developed from a cross between the Tanzanian farmer varieties, Namikonga and Albert. A one-step genetic linkage map comprising 943 SNP markers and 18 linkage groups spanning 1776.2 cM was generated. Phenotypic data from 240 F₁ progeny were obtained from two disease hotspots in Tanzania, over two successive seasons, 2013 and 2014. Two consistent QTLs linked to resistance to CBSD-induced root necrosis were identified in Namikonga on chromosomes II (qCBSDRNFc2Nm) and XI (qCBSDRNc11Nm) and a putative QTL on chromosome XVIII (qCBSDRNc18Nm). qCBSDRNFc2Nm was identified at Naliendele in both seasons. The same QTL was also associated with CBSD foliar resistance. qCBSDRNc11Nm was identified at Chambezi in both seasons, and was characterized by three peaks, spanning a distance of 253 kb. Twenty-seven genes were identified within this region including two LRR proteins and a signal recognition particle. In addition, two highly significant CMD resistance QTL (qCMDc12.1A and qCMDc12.2A) were detected in Albert, on chromosome 12. Both qCMDc12.1A and qCMDc12.2A lay within the range of markers reported earlier, defining the CMD2 locus. This is the first time that two loci have been identified within the CMD2 QTL, and in germplasm of apparent East African origin. Additional QTLs with minor effects on CBSD and CMD resistance were also identified.

     

Changes in the incidence and severity of Cassava mosaic virus disease, varietal diversity and cassava production in Uganda

A survey in 1994 assessed the incidence and severity of Cassava mosaic virus disease (CMD) in Uganda and the changes that had occurred since the previous survey in 1990–92. Three counties in each of 27 districts were selected and 15 fields were assessed per county. CMD occurred in each of the 1215 fields surveyed and the overall incidence within fields was 64% compared with 52% in the same districts in 1990–92. Mean incidence differed significantly between districts and between counties within some districts. The incidence and severity of CMD had increased in 20 and 23 of the 27 districts, respectively, compared with 1990–92, indicating the continued progress of the epidemic that was first reported in Uganda in 1988. In all but three districts, there had been a decrease in the total area of cassava. The number of different varieties that predominated in one or more fields had increased in 17 districts compared with 1990–92, although the total number of varieties encountered was little changed. Ebwanateraka was again the most widely grown variety but it was recorded less frequently and predominated or was co‐dominant in only two districts in 1994 compared with seven in 1990–92. The biggest decrease in the cultivation of Ebwanateraka had occurred in districts where there was a high incidence of CMD in 1990–92.     

Cassava brown streak disease: historical timeline,current knowledge and future prospects

Cassava is the second most important staple food crop in terms of per capita calories consumed in Africa and holds potential for climate change adaptation. Unfortunately, productivity in East and Central Africa is severely constrained by two viral diseases: cassava mosaic disease (CMD) and cassava brown streak disease (CBSD). CBSD was first reported in 1936 from northeast Tanzania. For approximately 70 years, CBSD was restricted to coastal East Africa and so had a relatively low impact on food security compared with CMD. However, at the turn of the 21st century, CBSD re‐emerged further inland, in areas around Lake Victoria, and it has since spread through many East and Central African countries, causing high yield losses and jeopardizing the food security of subsistence farmers. This recent re‐emergence has attracted intense scientific interest, with studies shedding light on CBSD viral epidemiology, sequence diversity, host interactions and potential sources of resistance within the cassava genome. This review reflects on 80 years of CBSD research history (1936–2016) with a timeline of key events. We provide insights into current CBSD knowledge, management efforts and future prospects for improved understanding needed to underpin effective control and mitigation of impacts on food security.     

Vector transmission of Banana streak virus in the screenhouse in Uganda

Although mealybug transmission of Banana streak virus.(BSV) by Planococcus citri and Saccharicoccus sacchar has been demonstrated elsewhere, these mealybugs have not been identified on bananas in Uganda and their role and that of other agents in BSV transmission is not well documented. Insect samples were collected from banana farms in sites with low, moderate and high BSV infections in Uganda. Subsequently, live mealybugs and aphids were again collected and used in acquisition, retention and transmission tests, and BSV diagnosed using TAS‐ELISA. Dysmicoccus brevipes (pineapple mealybug), S. sacchari (sugarcane mealybug) and Pentalonia nigronervosa (banana aphid) were the most abundant insect species from banana fields sampled. Abundance of D. brevipes was positively and significantly correlated with BSV incidence unlike that of. P. nigronervosa. Transmission studies in the screenhouse showed that mealybugs acquired BSV one day after feeding on virus sources and approached optimum acquisition after the third day. Pineapple and sugarcane mealybugs retained BSV up to 5 days from the day of transfer from the virus source. BSV was first detected in the recipient banana plants 4 wk after transmission using pineapple mealybug and 6 wk after inoculation using sugarcane mealybug. Under screenhouse conditions, both mealybugs therefore appear to transmit BSV semipersistently.     

Detection of Banana streak virus in field samples of bananas from Uganda

Banana streak virus (BSV) is one of the major constraints to banana production in Uganda. To develop a diagnostic technique, 59 samples were taken from 30 farms at 14 locations across Uganda; a further three samples were taken from infector plants for BSV epidemiology experiments. BSV was found in 51 of the field samples and in the three infector plants. The possible variation of the virus was assessed by serology (ISEM and ELISA) using a broad‐spectrum antiserum and by PCR. Virus was poorly detected in many of the samples by serological tests even though other techniques showed its presence. Virus was detected in most samples by PCR with a degenerate primer set on extracted viral DNA and on immune‐captured (1C) or directly bound (DB) virus particles. The epidemiology experiment samples did not give a product with these degenerate primers but did with other primer sets. A diagnostic procedure was developed involving concentrating the virus in sap by polyethylene glycol precipitation followed by 1C‐ or DB‐PCR using a degenerate primer set which detected virus in most samples.     

Disease spread patterns of Banana streak virus in farmers, fields in Uganda

Three surveys were conducted to establish the disease spread patterns of Banana streak virus (BSV) in farmers, fields in Uganda. Transects were traced both across the fields and from infection foci within a field. BSV incidence in adjacent quadrats was also determined to quantify statistically the spatial relationships of infected plants in the fields. Severity assessment along transects across fields revealed clusters of plants with moderate to high severity and clusters of plants with no BSV or low severity. Symptom severity decreased away from foci of infection (b=?0.014; P=0.0081). Observed frequency of infected quadrat counts differed from corresponding expected frequency of infected quadrat counts (Poisson, s distribution, x²; P<0.01). BSV– infected plants, therefore, were aggregated in well‐established fields. Aggregation of infected plants in farmers, fields and the decrease of severity away from infection foci suggest the likely involvement of a slow moving vector in BSV transmission.     

RNAi-mediated resistance to Potato spindle tuber viroid in transgenic tomato expressing a viroid hairpin RNA construct

Because of their highly ordered structure, mature viroid RNA molecules are assumed to be resistant to degradation by RNA interference (RNAi). In this article, we report that transgenic tomato plants expressing a hairpin RNA (hpRNA) construct derived from Potato spindle tuber viroid (PSTVd) sequences exhibit resistance to PSTVd infection. Resistance seems to be correlated with high-level accumulation of hpRNA-derived short interfering RNAs (siRNAs) in the plant. Thus, although small RNAs produced by infecting viroids [small RNAs of PSTVd (srPSTVds)] do not silence viroid RNAs efficiently to prevent their replication, hpRNA-derived siRNAs (hp-siRNAs) appear to effectively target the mature viroid RNA. Genomic mapping of the hp-siRNAs revealed an unequal distribution of 21- and 24-nucleotide siRNAs of both (+)- and (–)-strand polarities along the PSTVd genome. These data suggest that RNAi can be employed to engineer plants for viroid resistance, as has been well established for viruses.     

Clinostating effects on Apogee wheat resistance to wheat streak mosaic virus

The effects of vertical and horizontal clinostating with container velocity 2 rpm and platform velocity 1 rpm on Apogee wheat resistance to wheat streak mosaic virus (WSMV) were studied. For the first time the yield of grain was obtained from both, healthy and the WSMV-infected wheat plants under conditions of simulated microgravity (clinorotation) which is accounted for by the inclusion of various mechanisms in the process of plant adaptation to the factors of stresses. For the first time the stages of viral infection development were elucidated under the conditions of prolonged clinostating and the presence of the WSMV was detected in wheat plants on the 18th day after inoculation employing the method of polymerase chain reaction (PCR). In the test variant with vertical clinostating (R=1,0) the least favorable conditions for viral reproduction were identified. In the same variant the increases of the total nitrogen content in the ears were observed which may be an evidence of interferon-like protein synthesis in plant similar to the interferon system functioning in animals.     

Development of PCR markers linked to resistance to wheat streak mosaic virus in wheat

Wheat streak mosaic virus (WSMV), vectored by the wheat curl mite (Acer tulipae), is an important disease of wheat (Triticum aestivum L.) in the North American Great Plains. Resistant varieties have not been developed for two primary reasons. First, useful sources of resistance have not been available, and second, field screening for virus resistance is laborious and beyond the scope of most breeding programs. The first problem may have been overcome by the development of resistance to both the mite and the virus by the introgression of resistance genes from wild relatives of wheat. To help address the second problem, we have developed polymerase chain reaction (PCR) markers linked to the WSMV resistance gene Wsm1. Wsm1 is contained on a translocated segment from Agropyron intermedium. One sequence-tagged-site (STS) primer set (WG232) and one RAPD marker were found to be linked to the translocation containing Wsm1. The diagnostic RAPD band was cloned and sequenced to allow the design of specific PCR primers. The PCR primers should be useful for transferring Wsm1 into locally adapted cultivars.This is Journal Series No. J-4041 of the Montana Agricultural Experiment Station     

Temporal spread of cassava mosaic virus disease in a range of cassava cultivars in different agro-ecological regions of Uganda

The spread of cassava mosaic disease (CMD) in a range of cassava cultivars was studied in experiments and on-farm trials in different agro-ecological regions of Uganda in 1989–1990 and 1990–1991. No spread occurred in either experiment at the southernmost site near Kampala, but there was considerable spread at the four sites elsewhere and also in the on-farm trials in Luwero district. There were significant differences in the final incidence of disease between locations and between cultivars at each location. Where spread occurred it was more rapid in the Ugandan cvs Ebwanateraka, Senyonjo and Bao than in four of the five improved TMS cultivars introduced from Nigeria. These usually showed an apparent decline in incidence of CMD after reaching maxima 4 to 8 months after planting (MAP). The areas under the disease progress curves (AUDPCs) differed significantly between locations and cultivars and were less for cvs TMS 30572, TMS 30395, TMS 30337 and TMS 60142 than for cvs Ebwanateraka, Senyonjo, Bao and TMS 30786. Overall, the mean AUDPCs were greatest at Migyera in Luwero district in 1989–1990 and at Kagando in Kasese district in 1990–1991. They were significantly less at Mubuku in Kasese district in 1989–1990 than at the other two experimental sites where spread occurred. Adult whitefly vector populations were highest at Migyera and Kagando in the 1989–1990 and 1990–1991 trials, respectively, and they were higher on cvs Bao, Ebwanateraka and TMS 30786 than on other varieties. Mean numbers of adults increased until 3–5 MAP and then declined, but CMD incidence increased progressively to reach maxima at or near crop maturity. Locations with the largest numbers of adults also had a relatively high incidence of CMD. Symptoms of CMD were usually more severe on cvs Ebwanateraka, Bao and Bukalasa 11 than on the TMS cultivars, on which symptoms remained slight throughout growth and usually decreased from 5 MAP. The differences between sites, the resistance of the cultivars and the relationship between CMD incidence and whitefly populations are discussed.     

Broad virus resistance in transgenic plants

Viruses are significant threats to agricultural crops worldwide and the limited sources of natural resistance warrant the development of novel resistance sources. Several methods of transgenic protection have been successfully applied, including protein- and RNA-mediated approaches. Increased understanding of the molecular biology of virus infection is starting to bear fruit, enabling specific strategies to be designed for virus resistance in crops.