Control of brown patch (Rhizoctonia solani) in tall fescue (Festuca arundinacea Schreb.) by host induced gene silencing.

KEY MESSAGE: Transgenic tall fescue plants expressing RNAi constructs of essential genes of Rhizoctonia solani were resistant to R. solani. Tall fescue (Festuca arundinacea Schreb.) is an important turf and forage grass species widely used for home lawns and on golf courses in North Carolina and other transition zone states in the US. The most serious and frequently occurring disease of tall fescue is brown patch, caused by a basidiomycete fungus, Rhizoctonia solani. This research demonstrates resistance to brown patch disease achieved by the application of host induced gene silencing. We transformed tall fescue with RNAi constructs of four experimentally determined "essential" genes from R. solani (including genes encoding RNA polymerase, importin beta-1 subunit, Cohesin complex subunit Psm1, and a ubiquitin E3 ligase) to suppress expression of those genes inside the fungus and thus inhibit fungal infection. Four gene constructs were tested, and 19 transgenic plants were obtained, among which 12 plants had detectable accumulation of siRNAs of the target genes. In inoculation tests, six plants displayed significantly improved resistance against R. solani. Lesion size was reduced by as much as 90 %. Plants without RNAi accumulation did not show resistance. To our knowledge, this is the first case that RNAi constructs of pathogen genes introduced into a host plant can confer resistance against a necrotrophic fungus.

Maize Lethal Necrosis (MLN), an Emerging Threat to Maize-Based Food Security in Sub-Saharan Africa.

In sub-Saharan Africa, maize is a staple food and key determinant of food security for smallholder farming communities. Pest and disease outbreaks are key constraints to maize productivity. In September 2011, a serious disease outbreak, later diagnosed as maize lethal necrosis (MLN), was reported on maize in Kenya. The disease has since been confirmed in Rwanda and the Democratic Republic of Congo, and similar symptoms have been reported in Tanzania, Uganda, South Sudan, and Ethiopia. In 2012, yield losses of up to 90% resulted in an estimated grain loss of 126,000 metric tons valued at $52 million in Kenya alone. In eastern Africa, MLN was found to result from coinfection of maize with Maize chlorotic mottle virus (MCMV) and Sugarcane mosaic virus (SCMV), although MCMV alone appears to cause significant crop losses. We summarize here the results of collaborative research undertaken to understand the biology and epidemiology of MLN in East Africa and to develop disease management strategies, including identification of MLN-tolerant maize germplasm. We discuss recent progress, identify major issues requiring further research, and discuss the possible next steps for effective management of MLN.

Characterization of the 3' proximal gene of the citrus tristeza closterovirus genome.

The 3' proximal open reading frame (ORF 11) in the citrus tristeza virus (CTV) genome potentially encodes a protein of 209 amino acids with an estimated molecular weight of 23 kDa (p23). The p23 ORF from the severe Florida strain T36 of CTV was expressed in Escherichia coli, and the expressed protein was used to raise polyclonal antibodies in a rabbit. Using these antisera on a Western blot, a protein of expected size (23 kDa) was detected in tissue extracts from CTV-infected citrus but not from uninfected citrus. Most of the p23 protein was found in the soluble, cytoplasmic fraction. Comparison of the sequence of p23 genes from several biologically and geographically diverse CTV isolates indicated a high degree of conservation for this gene and for the RNA binding motif in particular. A cluster dendrogram of the deduced amino acid sequences correlated with the biological properties of the isolates, forming distinct groups of mild, quick decline on stem pitting-inducing isolates. Therefore it is possible that, in addition to the capsid protein gene, the p23 gene also may serve as an indicator for disease severity.

Progress on strain differentiation of Citrus tristeza virus and its application to the epidemiology of citrus tristeza disease.

Citrus tristeza virus (CTV) occurs in most citrus producing regions of the world, and it is the most serious viral pathogen of citrus. With the recent establishment of the brown citrus aphid, Toxoptera citricida, its most efficient vector, on Madeira Island (Portugal) and in Florida (USA) and the countries of the Caribbean Basin, the impact of CTV is likely to increase in these regions. Since there are many strains of CTV and CTV infections frequently occur as mixtures of several strains, it is necessary to be able to distinguish the strains for regulatory purposes, disease management and epidemiology. We describe the evolution of techniques developed to detect CTV and to differentiate the individual strains, and present the results of tests using these latest methods on CTV isolates from mainland Portugal, Madeira Island and Florida. Mild and decline-inducing strains of CTV were detected in mainland Portugal and mild, decline-inducing and severe stem pitting strains on Madeira Island. In Florida we demonstrated the presence of infections that reacted with probes made against stem pitting strains not previously detected there. It is concluded that CTV presents a significant threat to citrus production in mainland Portugal, on Madeira Island and in the neighbouring countries of the Mediterranean Basin, as well as in Florida, elsewhere in the USA and throughout the Caribbean Basin, especially following the widespread establishment of T. citricida throughout the region.

Potyviridae: genus Rymovirus.

The genus Rymovirus of the family Potyviridae is comprised of seven rod-shaped viruses with the shared characteristic of being transmitted by mites. Aside from this distinguishing feature, rymoviruses are similar to aphid-transmitted potyviruses in that they share a similar particle morphology, some similar antigenic determinants, similar physico-chemical properties, the ability to induce the formation of cytoplasmic cylindrical inclusions, and the ability to infect only graminaceous hosts. In vitro translation studies with wheat streak mosaic virus (WSMV) suggest that this rymovirus uses a potyviral proteolytic processing strategy to express the 3' terminal capsid protein. At the molecular level, limited nucleotide sequence data for WSMV show similarities with aphid-transmitted potyviruses in the potyviral capsid protein, large nuclear inclusion and cylindrical inclusion regions. Thus, given the similarities between the rymoviruses and the potyviruses, it is appropriate to include this genus within the family Potyviridae.

Identification to the species level of the plant pathogens Phytophthora and Pythium by using unique sequences of the ITS1 region of ribosomal DNA as capture probes for PCR ELISA.

The ribosomal internal transcribed spacer 1 region was sequenced for 10 species of Pythium and eight species of Phytophthora. Alignment of the sequences revealed considerable sequence microheterogeneity, which was utilized to prepare a capture probe of unique sequence for each species. The capture probes were tested by PCR ELISA, combining the sensitivity and specificity of the polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). The probes were entirely species specific, enabling the detection and identification of the amplified DNA of species from individual cultures or from mixed samples of the DNAs of two different species. This approach to species identification, which provides a molecular technology to process large numbers of samples and still identify the fungi with a high level of confidence, may greatly reduce the resources and the time of highly trained specialists currently needed to identify these important species of plant pathogenic fungi.

Development of a dot-immunobinding assay for detection of citrus tristeza virus.

The dot-immunobinding assay (DIBA) was adapted for detection of citrus tristeza virus (CTV) and compared with DAS-ELISA and DAS-indirect ELISA. DIBA was easy to perform and as sensitive as either ELISA procedure for CTV diagnosis. The entire test could be performed in 2-3 h using polyclonal antibodies, with minimal laboratory equipment. Three different polyclonal antibodies gave a strong positive reaction with 12 selected CTV isolates; however, each serum had to be cross-absorbed with sap from healthy plants before use. The broad spectrum 3DF1 monoclonal antibody reacted with most of the CTV isolates. The MCA-13 strain-specific monoclonal antibody was specific for most severe CTV isolates. As blocking agents, 3% bovine serum albumin (BSA), 3% gelatin, 0.5% non-fat dry milk or 5% Triton X-100 gave an adequate white background on the nitrocellulose membranes and permitted discrimination between infected and healthy samples. However, 3% gelatin gave the best contrast between green for the healthy samples, and purple color for infected samples.

A polymerase chain reaction method adapted for selective amplification and cloning of 3' sequences of potyviral genomes: application to dasheen mosaic virus.

'Universal' degenerate oligonucleotide primers were used to amplify cDNA sequences containing the 3' untranslated region (3' UTR) and a portion of the coat protein gene sequence of dasheen mosaic potyvirus (DMV). These primers were based on the conserved WCIEN and QMKAAA 'boxes' of the potyviral coat protein and the poly-A tail found at the 3' end of the genome. The forward genome-sense primers were designed taking into consideration the codon degeneracy of the WCIEN and QMKAAA residues for several potyviruses. The anti-sense reverse primer has 21 T residues followed by either A, C or G at the 3' end to ensure specific priming at the end of the 3' UTR and beginning of the poly-A tail. The specificity of amplification was verified using the known potyviruses (watermelon mosaic 2 and soybean mosaic viruses). To demonstrate the applicability of this method, the 3' UTR of the unsequenced DMV was amplified, cloned and sequenced. Sequence comparisons with other potyviral 3' UTRs revealed DMV to be quite distinct: nucleotide sequence similarities of only 34% to 44% were found with sequenced viruses indicating no close affinities with any other potyvirus. The potyvirus 3' sequence amplification procedure is simple and rapid, is potentially useful in developing virus specific probes and may be used to differentiate strains and species of potyviruses on the basis of the 3' UTR sequences.

Distribution and Characterization of Citrus tristeza virus in South Florida Following Establishment of Toxoptera citricida.

The incidence of Citrus tristeza virus (CTV) was found to increase significantly in southern Florida within 2 years after the establishment of its most efficient vector, Toxoptera citricida (Kirkaldy). Increased incidence of both mild and severe strains was documented, with the incidence of severe strains increasing more than mild strains. Molecular probes capable of differentiating mild, quick decline and various types of stem-pitting strains demonstrated that trees often were infected with more than one strain of CTV, with trees containing up to five different strains. Some CTV strains detected in the southeast urban corridor of Florida and in commercial groves in southwest Florida were found to react with probes specific for stem-pitting strains known from elsewhere in the world. The implications of the presence of these CTV strains in Florida and their possible presence in citrus budwood scion trees are discussed.

In vivo and in vitro expression analysis of the RNA-dependent RNA polymerase of Citrus tristeza virus.

Expression of the RNA-dependent RNA polymerase (RdRp) of Citrus tristeza virus (CTV) was studied in vivo and in vitro using a polyclonal antiserum raised against the recombinant CTV-RdRp protein. Although a 57-kDa CTV-RdRp was expected to be expressed by a +1 translational frameshift at the carboxyl terminus of a 400-kDa polyprotein, a 50-kDa protein was detected in CTV-infected but not in healthy citrus tissue by Western blot. This suggests that the RdRp was cleaved from the CTV polyprotein. The 50-kDa protein was present in both the cytoplasmic and membrane fractions, but it accumulated mainly in the membrane fraction, where most of the replication-associated proteins of RNA viruses are found. When the expression of a cloned CTV-RdRp gene encoding a 60-kDa fusion protein was studied in vitro in a rabbit reticulocyte lysate system, two smaller proteins of about 50 kDa and 10 kDa were detected in addition to the expected 60-kDa protein. All three proteins were immunoprecipitated with the anti-CTV-RdRp serum, suggesting that the 50-kDa and 10-kDa proteins were fragments of the 60-kDa CTV-RdRp fusion protein. When the expression of the RdRp was analyzed at different times during in vitro translation, the 60-kDa and 50-kDa proteins were detected at all time points, and a small amount of the 10-kDa protein was detected after 30 min of translation. These results suggest that the CTV-RdRp may also be cleaved in vitro in the rabbit reticulocyte lysate.

Application of recombinant DNA technology to plant protection: molecular approaches to engineering virus resistance in crop plants.

Developments in plant tissue culture, plant transformation and regeneration, and improvements in techniques to isolate and manipulate viral genes have led to the exploitation of the concept of 'cross protection': turning the virus onto itself and controlling it with its own genes. By introducing and expressing genes of viral origin in crop plants, scientists have engineered resistance to several plant viruses. Some of the approaches, used singly or in combination, include expression of viral-coat protein, untranslatable sense or antisense RNA, satellite RNA, virusspecific 'neutralizing' antibody genes, plant viral replicase, protease or movement proteins and defective, interfering RNA. All of these approaches have resulted in manifestation of virus resistance to varying degrees in several commercially important crop plants. This review summarizes the recent advances in engineering virus resistance using the above approaches, and lists specific examples of their use in cultivated crop plants of economic importance.

Asparate transcarbamylase activity in etiolated cowpea hypocotyls treated with 2,4-dichlorophenoxyacetic Acid.

Treating etiolated cowpea (Vigna unguiculata) seedlings with 2,4-dichlorophenoxyacetic acid resulted in 2.5-, 3.9-, and 6.5-fold increases in DNA, soluble protein, and RNA, respectively, over untreated controls 84 hours after treatment. Aspartate transcarbamylase activity increased within 12 hours after treatment, and by 84 hours it was almost 12-fold greater than that in the untreated controls. During that time, activity in untreated controls dropped 60%. The assay used (14)C-aspartate, which was then separated from the (14)C-ureidosuccinate product by Dowex 50 (H(+) form) column chromatography. Thin layer chromatography of the reaction product indicated that most of the carbamyl-phosphate-dependent radioactivity co-chromatographed with ureidosuccinate. The reaction has a pH optimum near 10.0 and is inhibited by uridine 5'-phosphate and succinate. The data suggest that aspartate transcarbamylase is important in pyrimidine biosynthesis in 2,4-dichlorophenoxyacetic acid-treated seedlings.

Effects of inhibitors of RNA and protein synthesis on aspartate transcarbamylase activity in etiolated plant tissue.

Aspartate transcarbamylase (ATCase) activity declines in etiolated cowpea (Vigna unguiculata L. Walp.) and soybean (Glycine max L. Merr.) hypocotyls between 3 and 11 days after planting. Treating cow-pea hypocotyls with cycloheximide (CH), actinomycin D (AMD), 6-methyl purine (6-MP), or cordycepin increases ATCase activity up to 740, 350, 465, and 305%, respectively, over water-treated controls 48 to 72 hours after treatment. In contrast erythromycin had no effect, and d-threo-chloramphenicol (CHL) reduced ATCase activity nearly 40%. CH, AMD, and CHL, whose effects were further characterized, each markedly reduced total RNA synthesis and protein synthesis. Respiration was stimulated by CH and AMD and reduced by CHL. In soybean, CHL-treated tissues and water-treated controls had comparable ATCase activities 48 hours after treatment, while AMD, 6-MP, and CH treatments reduced activities 29, 37, and 78%, respectively. The results suggest that the level of ATCase activity in etiolated cowpea hypocotyls is regulated by a mechanism or mechanisms that are interfered with by inhibition of RNA and protein synthesis. Possibly the mechanism is absent from etiolated soybean hypocotyls.

Variability in the length of the amino terminal sequence contributes to the capsid protein diversity among dasheen mosaic potyvirus isolates.

Western blot analysis of several dasheen mosaic virus isolates revealed molecular weight differences among their capsid proteins (CPs). Sequence analysis of the CP gene showed that the CP of isolate TEN was 15 amino acids shorter than that of isolate LA due to a 57-base deletion and a 12-base insertion into the 5' end of the TEN CP gene. Our data suggest that frequent deletions and insertions are responsible for the CP size diversity among DMV isolates.

Unusual amino-terminal sequence repeat characterizes the capsid protein of dasheen mosaic potyvirus.

The 3'-terminal region of a Florida isolate of dasheen mosaic potyvirus (DMV-LA) genome including the coat protein (CP) gene was cloned and sequenced. Protease digestion was predicted to occur between the glutamine and alanine residues at positions 79 and 80 of the 408 residue long polypeptide to produce a CP of 329 amino acids with an estimated M(r) of 36,229. Following the putative protease recognition site is a DAG sequence, which is conserved among aphid-transmitted potyviral CPs. There is an unusual and unique stretch of 52 amino acids after the DAG that is repetitive and rich in threonine and asparagine. A sequence of 10 residues (GNNTNTNTSNT) was repeated three times in tandem within this stretch and was followed by six proline residues. Several potential glycosylation sites were found clustered within this region. Expression in Escherichia coli and Western blotting of the CP confirmed its size and serological identity. Sequence comparisons and phylogenetic reconstructions indicated that DMV is a distinct potyvirus within the passionfruit woodiness virus subgroup cluster.

Tomato DNA contains no detectable regions complementary to potato spindle tuber viroid as assayed by solution and filter hybridization.

A previous report (Hadidi et al. (1976) Proc. Nat. Acad. Sci. USA73, 2453-2457) has suggested that potato spindle tuber viroid (PSTV) RNA contains sequences complementary to the DNA of its host and, to a lesser extent, to the DNA of some nonhost species. Our studies indicate, however, that the apparent hybridization between (125)I-labeled PSTV and tomato DNA which can be observed in both solution and filter hybridization experiments is a consequence of the hybridization of host RNAs (mostly ribosomal) which are minor contaminants of the polyacrylamide gel-purified viroid preparations. When the level of these contaminants is reduced by further purification of the viroid in gels containing 8 M urea, no hybridization to tomato DNA could be detected. Thus, if sequence homology exists, it must be very minimal and its detection is beyond the capabilities of the methods employed.

Characterization of grapefruit plants (Citrus paradisi Macf.) transformed with citrus tristeza closterovirus genes.

Grapefruit (Citrus paradisi Macf. cv Duncan) plants were transformed with several sequences from citrus tristeza closterovirus (CTV) that varied in terms of position in the CTV genome and virus strain origin in an attempt to obtain resistant plants. The sequences included the capsid protein gene from three different strains, a nontranslatable version of the capsid protein gene, the replicase (RdRp), the minor capsid protein (p27), a highly transcribed gene of unknown function (p20) and the more conserved 3' end of the genomic RNA. Transgenic plants were generated from all of the constructs, except from the p20 and p27 genes. Southern and Western blot analyses demonstrated that stably transformed grapefruit plants were obtained and that at least some transgenes were expressed. In a first effort at virus challenge, 25 transgenic lines were graft inoculated with a severe strain of CTV. Although some transgenic plants averaged lower titers of virus than controls, there was great variability in titer in both controls and transgenic plants, and all were apparently susceptible to the virus.

Characterization of citrus tristeza virus subgenomic RNAs in infected tissue.

Citrus tristeza virus (CTV) specific RNAs extracted from infected citrus tissue were analyzed by Northern blot hybridization. RNAs were characterized by size and identified using cDNA probes specific to nine open reading frames (ORFs) identified by the analysis of sequence obtained from cDNA clones of the T36 isolate of CTV. Sequence specific cDNA probes identified the genomic RNA as well as subgenomic RNAs representing the p33, p65, p61, p27, p25, p18, p13, p20, and p23 ORFs in extracts of total or double-stranded RNA (dsRNA) isolated from infected tissue. A probe derived from the 3' terminal ORF (p23) hybridized to each of these subgenomic RNAs, indicating that the RNAs are 3' coterminal. The relative amounts of the different subgenomic RNAs varied widely. The RNAs for the p20 and p23 ORFs were the most abundant and surpassed the amount of the p25 or capsid protein specific subgenomic RNA. The number and sizes of the CTV subgenomic RNAs were the same in total RNA and dsRNA preparations. Propagation of T36 in seven different citrus hosts did not alter the pattern of subgenomic RNAs.

Molecular characterization of a structural epitope that is largely conserved among severe isolates of a plant virus.

Direct molecular evidence was obtained for the critical role of a single amino acid residue in a structural epitope distinguished by the monoclonal antibody MCA-13, which reacts selectively with severe isolates of citrus tristeza virus (CTV). Different CTV isolates cause a wide range of symptoms in the diverse citrus species they affect. Severe symptoms include decline, stem pitting, and seedling yellows. Plants infected by mild isolates are essentially symptomless. The monoclonal antibody MCA-13, which discriminates severe isolates from mild isolates of the virus, was used to map its epitope on the coat protein of CTV. A diverse group of coat protein genes of geographically and biologically distinct CTV isolates which are either MCA-13-reactive or MCA-13-nonreactive was cloned and sequenced. A series of mutant coat protein genes was constructed through oligonucleotide-directed, site-specific mutagenesis. The reactivity of the wild-type and mutant coat proteins expressed in Escherichia coli was evaluated by Western blotting using MCA-13 and polyclonal antibody prepared to CTV-coat protein. A single nucleotide alteration resulting in a Phe-->Tyr mutation at position 124 of the coat protein abolished the MCA-13 reactivity of a severe isolate, whereas a Tyr-->Phe mutation at the same site conferred MCA-13 reactivity on the coat protein of a previously nonreactive mild isolate of CTV.