Inducible Resistance to Maize Streak Virus

Maize streak virus (MSV), which causes maize streak disease (MSD), is the major viral pathogenic constraint on maize production in Africa. Type member of the Mastrevirus genus in the family Geminiviridae, MSV has a 2.7 kb, single-stranded circular DNA genome encoding a coat protein, movement protein, and the two replication-associated proteins Rep and RepA. While we have previously developed MSV-resistant transgenic maize lines constitutively expressing “dominant negative mutant” versions of the MSV Rep, the only transgenes we could use were those that caused no developmental defects during the regeneration of plants in tissue culture. A better transgene expression system would be an inducible one, where resistance-conferring transgenes are expressed only in MSV-infected cells. However, most known inducible transgene expression systems are hampered by background or “leaky” expression in the absence of the inducer. Here we describe an adaptation of the recently developed INPACT system to express MSV-derived resistance genes in cell culture. Split gene cassette constructs (SGCs) were developed containing three different transgenes in combination with three different promoter sequences. In each SGC, the transgene was split such that it would be translatable only in the presence of an infecting MSV’s replication associated protein. We used a quantitative real-time PCR assay to show that one of these SGCs (pSPLITrep^III-Rb-Ubi) inducibly inhibits MSV replication as efficiently as does a constitutively expressed transgene that has previously proven effective in protecting transgenic maize from MSV. In addition, in our cell-culture based assay pSPLITrep ^III-Rb-Ubi inhibited replication of diverse MSV strains, and even, albeit to a lesser extent, of a different mastrevirus species. The application of this new technology to MSV resistance in maize could allow a better, more acceptable product.     

Incidence and infection rate of Maize streak virus disease by Cicadulina triangular on maize plants and its distribution from the lowest diseased leaf under tropical conditions

Several programmes have been initiated for the development of maize varieties with resistance traits of Maize streak virus (MSV) by International Institute of Tropical Agriculture (IITA), Ibadan, and have been released to farmers and research scientists. Therefore, a survey was conducted in five states in the south west of Nigeria (Oyo, Ogun, Ondo, Ekiti and Osun) during the raining planting season to determine the incidence of MSV disease by visual examination and sero-diagnostic screening of symptomatic plants. The determination of infection rate of MSV disease by Cicadulina triangular on maize plant and its distribution from the lowest diseased leaf was also studied. The mean MSV disease incidence observed in these states was 35.95% which confirms the presence of MSV in the south west of Nigeria. Sero-diagnostic screening of virus-induced symptomatic leaf samples indicated that out of the 250 leaves sampled per state, 24.4% tested positive for MSV in Oyo, 25.6% in Ondo, 34% in Ogun, 19.6% in Ekiti and 38.8% in Osun. In two-week-old plants, symptoms developed on the leaves that were emerging at the time of inoculation, while in six-week-old plants, symptoms developed on the leaves directly below the emerging leaves irrespective of the number of C. triangular used. These suggest that the lowermost leaf with symptoms of the disease indicates the growth stage at which a plant was infected. There was a relationship between symptom expression and plant age which could be very effective when carrying out surveys to gather information for epidemiological studies. In addition, the 10 varieties of maize inoculated with MSV through C. triangular transmission showed no significant difference in disease severity over time irrespective of the number of C. triangular used.     

Maize seed selection by East African smallholder farmers and resistance to Maize streak virus*

Interviews identified that most small‐scale maize farmers in central Uganda and in the Southern Highlands of Tanzania plant home‐saved seed of landraces or seed derived from various open‐pollinated and hybrid varieties. Some farmers also bought a portion of their seed, either certified seed, locally traded seed or even maize sold for consumption. Selection for home‐saved seed was generally among harvested cobs. Big cobs with many, regularly arranged, large, white, flint kernels were preferred. A maize cob may bear several hundred seeds, so a farmer needs to save <1% of cobs for seed. A form of resistance in which plants show only moderate symptoms and suffer only a small reduction in yield when infected has been incorporated in some released varieties. Because not all plants in most crops are infected and because plants uninfected with Maize streak virus (MSV) tend to produce bigger cobs than infected resistant plants, the few cobs selected by a farmer for seed may all be from the uninfected ‘escapes’, with no preferential selection of resistant types. On‐station simulation of the farmers’ selection process in a crop of the MSV‐resistant maize variety, Longe 1, confirmed this. An alternative very strong form of MSV resistance was identified.     

Assessment of yield losses as a result of co-infection by maize streak virus and maize stripe virus in Mauritius

The effect of co-infection by maize streak virus (MSV) and maize stripe virus (MStV) on plant growth and grain yield was investigated in a susceptible variety of maize (Zea mays), ZS 5206, in Mauritius. Under natural conditions MSV, transmitted by the leafhopper Cicadulina mbila, was normally established before MStV, which is vectored by the planthopper Peregrinus maidis; as a result, MStV symptoms were often partially or completely masked by those of MSV, making MStV detection by symptomatology very unreliable. MSV and MStV were diagnosed by ELISA and MStV by a novel method of detecting the MStV-coded non-capsid protein. The maize hybrid ZS 5206 was inoculated with either MSV, MStV or both, at two stages in the growth cycle (3–5 or 7–10 leaf stage). A greater reduction in plant growth was observed in plants inoculated singly with MStV (80% and 29% for first and second stage, respectively) than with MSV (50% and 23%, respectively). No cobs were produced by plants singly infected with MStV at the first stage, or co-infected with MSV and MStV at both stages; however, marginal grain production was recorded in plants singly infected with MSV at the first stage (91% reduction), or infected either with MSV or MStV, at the second stage (65% and 80% reduction, respectively). In maize hybrid ZS 5206, MStV is more virulent than MSV; co-infection by both viruses causes greater reductions in plant growth and grain yield than single infection by either virus at a given stage of plant development. In the event of co-infection by MSV and MStV, yield losses can be erroneously attributed to MSV only if the symptoms of MStV are masked by those of the former and if adequate methods for MStV detection are not used.     

Detection of a non-structural protein of M r 11 000 encoded by the virion DNA of maize streak virus

A polypeptide of approximately 11 000 daltons (11 kDa protein) encoded by an open reading frame (10.9 ORF) from the virion sense of maize streak virus (MSV) DNA has been detected among the products of in vitro translation reactions programmed with RNA from infected maize plants and also in total protein extracts from infected leaves. The 11 kDa protein has not been detected in virions and is therefore proposed to have a nonstructural role.Viral DNA with an additional in-frame translation stop codon in the 10.9 ORF was not infectious when transmitted to maize plants via Agrobacterium tumefaciens agroinfection, suggesting that the 10.9 ORF may be essential for virus function. Computer comparison data show that equivalent ORFs in wheat dwarf virus (WDV) and digitaria streak virus (DSV) have some sequences in common with the 10.9 ORF of MSV. Further-more, the absence of similar sequences in geminiviruses which infect dicotyledonous plants suggests that the 11 kDa protein and its putative homologs in WDV and DSV have a function necessary only for those geminiviruses which infect the Gramineae.The significance of the 11 kDa protein in relation to expression of the virion sense DNA of MSV is discussed.     

Viruses and thyroiditis: an update

Background

Despite the demonstration that geminiviruses, like many other single stranded DNA viruses, are evolving at rates similar to those of RNA viruses, a recent study has suggested that grass-infecting species in the genus Mastrevirus may have co-diverged with their hosts over millions of years. This "co-divergence hypothesis" requires that long-term mastrevirus substitution rates be at least 100,000-fold lower than their basal mutation rates and 10,000-fold lower than their observable short-term substitution rates. The credibility of this hypothesis, therefore, hinges on the testable claim that negative selection during mastrevirus evolution is so potent that it effectively purges 99.999% of all mutations that occur.

Results

We have conducted long-term evolution experiments lasting between 6 and 32 years, where we have determined substitution rates of between 2 and 3 × 10^-4 substitutions/site/year for the mastreviruses Maize streak virus (MSV) and Sugarcane streak Réunion virus (SSRV). We further show that mutation biases are similar for different geminivirus genera, suggesting that mutational processes that drive high basal mutation rates are conserved across the family. Rather than displaying signs of extremely severe negative selection as implied by the co-divergence hypothesis, our evolution experiments indicate that MSV and SSRV are predominantly evolving under neutral genetic drift.

Conclusion

The absence of strong negative selection signals within our evolution experiments and the uniformly high geminivirus substitution rates that we and others have reported suggest that mastreviruses cannot have co-diverged with their hosts.     

A single amino acid change in the coat protein of Maize streak virus abolishes systemic infection, but not interaction with viral DNA or movement protein

Functional coat protein (CP) is important for host plant infection by monopartite geminiviruses. We identified a proline-cysteine-lysine (PCK) motif at amino acids 180–182 of the maize streak virus (MSV) CP that is conserved in most of the cereal–infecting Mastreviruses. Substitution of the lysine (K) with a valine (V) in the CP of MSV to produce mutant MSVCP182V abolished systemic infection in maize plants, although the mutant replicated around the inoculation site and, unlike other MSV CP mutants, enabled single-stranded (ss) DNA accumulation in suspension cells. The stability of the mutant protein, CP182V, in infected cells was confirmed by immunoblotting, but virions could not be detected. Like the wild-type (wt) CP, CP182V localized to the nucleus when expressed in insect and tobacco cells, and the Escherichia coli-expressed protein bound both ss and double-stranded DNA and interacted with movement protein in vitro. Taken together, these data suggest that mutation of amino acid 182 affects virion formation of MSV, either by affecting encapsidation per se or by affecting particle stability, and that virions are necessary for the long-distance movement of MSV in maize plants.     

Comparison of transmission abilities of four Cicadulina species vectors of maize streak virus from Nigeria

Four Cicadulina species [Cicadulina arachidis China, Cicadulina dabrowskii Webb, Cicadulina mbila (Naude), and Cicadulina storeyi China (Homoptera: Cicadellidae)] found during field surveys in 1997–1999 across five ecological zones in Nigeria were reared in screenhouses and females were used in a study to compare their abilities to transmit Maize streak virus (Geminiviridae: genus Mastrevirus) from maize to maize using the susceptible variety Pool 16. The results showed that for both acquisition access feeding periods (AAP) and inoculation access feeding periods (IAP), transmission efficiencies decreased in the following order: C. storeyi > C. mbila > C. arachidis > C. dabrowskii. The transmission efficiencies of these vectors increased with longer feeding periods, as the means of susceptible test plants that showed Mastrevirus symptoms for both acquisition and IAPs were higher for 24 and 48 h than for 1 and 2 h for all four species studied. The epidemiological implications of these differences in transmission abilities are discussed.     

Specificity of Agrobacterium-mediated delivery of maize streak virus DNA to members of the Gramineae

Parameters affecting the efficiency of agroinfection of maize streak virus (MSV) in maize have been determined. Monomeric units, cloned at a number of sites in the MSV genome were not infectious but multimeric units containing partial duplications were equally as infectious as complete tandem dimeric clones. Inoculation of tandem dimeric units conjugated into different strains of Agrobacterium showed that both A. tumefaciens and A. rhizogenes were able to transfer DNA to maize and this ability was Ti (or Ri) plasmid-specific. Nopaline strains of A. tumefaciens and both agropine and mannopine A. rhizogenes strains efficiently transferred MSV DNA to maize. A number of strains were capable of MSV DNA transfer to other members of the Gramineae, providing information which may be essential for Agrobacterium-mediated transformation of monocotyledonous plants.     

A study of the mode of transmission of maize streak virus by Cicadulina mbila using an enzyme-linked immunosorbent assay

Two batches of Cicadulina mbila were given two distinct acquisition access periods (AAP) (3 h and 50 h) on maize plants infected with maize streak virus (MSV). Infectivity assays on susceptible maize were carried out 1, 3, 10, 17, 26 and 35 days after the AAP. Transmission efficiency was significantly higher for C. mbila subjected to the 50-h AAP. At the same time as the infectivity assays, the amount of MSV in each leafhopper was determined by an indirect double antibody sandwich (IDAS) ELISA. There were more ELISA-positive insects after the 50-h AAP than after the 3-h AAP. In the group given a 3-h AAP, only 7% of the insects tested between day 1 and 35 were found to be positive by ELISA. In contrast, after the 50-h AAP, the majority of C. mbila were positive, yet a decrease in ELISA-positive insects was noticed from day 17 onwards. Using a calibration curve obtained with purified virus, as little as 0.15 ng of MSV per insect could be measured by the IDAS-ELISA. A mean value of 0.36 ng of MSV per C. mbila was found 3 days after the 50–h acquisition, whereas 14 days later there was only 0.20 ng of virus per insect. For comparison, when leafhoppers were kept on infected maize, they displayed substantial accumulation of MSV up to an average of 3.83 ng of MSV per insect after 35 days of continuous acquisition. The amount of virus per insect detected in females was usually greater than the amount detected in males. Our results suggest that MSV does not multiply in C. mbila and contribute to the understanding of the persistence of transmission efficiency in the absence of virus multiplication.     

Comparison and characterization of maize stripe and maize line viruses

Two morphologically similar viruses isolated from maize in East Africa induced two distinct symptom types in maize. One, designated maize stripe virus (MSV), showed broad yellow stripes or a yellowing of the entire leaf, acute bending of the shoot apex and severe stunting. The second, maize line virus (MLV), induced continuous, narrow yellow lines along the leaf veins and did not cause apical bending or stunting. MSV and MLV were both transmitted by Peregrinus maidis (Delphacidae), but not by Cicadulina mbila (Jassidae) or by sap inoculation. Both viruses were purified by extracting systemically infected leaves in 0–5 M sodium citrate buffer and clarifying with 7 ml n-butanol/100 ml extract, followed by differential, and finally sucrose density gradient, centrifugation. Partially purified preparations of both viruses contained isometric viruslike particles of two sizes: MSV particles were 35 and 40 nm in diameter with sedimentation coefficients (so₂o, w) ^I09 ^anI0o respectively; MLV particles were 28 and 34 nm in diameter. Antisera prepared against MSV and MLV had dilution end points of 1/128 and 1/64 respectively in agar-gel diffusion tests. In tests with low-titre antisera, MSV did not react with MLV antiserum and MLV did not react with MSV antisreum; in the presence of antiserum containing antibodies to both MSV and MLV, the two viruses formed precipitin bands which crossed in the pattern of non-identity. Maize streak virus and maize mottle virus showed no serological relationship with MSV or MLV. On the basis of particle size and serology MSV and MLV are shown to be two distinct and possibly unrelated viruses. MSV and MLV apparently are dissimilar from any characterized viruses of the Gramineae.     

Transmission by Laodelphax striatellus Fallen of Rice black‐streaked dwarf virus from Frozen Infected Rice Leaves to Healthy Plants of Rice and Maize

Rice black‐streaked dwarf virus (RBSDV) is transmitted naturally to important crops such as rice, maize, barley and wheat in a persistent manner by the planthoppers, Laodelphax striatellus, Unkanodes sapporona and Unkanodes albifascia. Insect vector transmission tests are the basis for identifying viral incidence, evaluating the resistance of varieties and selecting resistance sources for rice and maize breeding. A simple, rapid and reliable method is described by which virus‐free small brown planthoppers (L. striatellus) acquired RBSDV from frozen infected rice leaves and transmitted it to healthy rice and maize plants. After feeding on frozen infected rice leaves, the planthoppers were tested by RT‐PCR for the presence of virus after 10, 15, and 22 days, respectively. The percentages of RBSDV‐containing insects were 0, 25 and 71.43% of L. striatellus fed on frozen infected rice leaves compared to 0, 28.25 and 71.43% of L. striatellus fed on fresh infected rice leaves, respectively. In transmission tests, three of eight rice seedlings (37.5%) and four of eight maize seedlings (50%) were inoculated by the planthoppers that had fed previously on frozen leaves and had allowed a 22 days latent period and showed typical disease symptoms. As a positive control, four of eight rice seedlings (50%) and four of six maize seedlings (66.67%) became infected. All rice and maize plants expressing disease symptoms were identified as virus‐positive by RT‐PCR. These results indicated that the planthoppers acquired RBSDV from frozen infected leaves and transmitted the virus to healthy plants.     

Cytopathology and Ultrastructure of Some African Isolates of Maize Streak and Sugarcane Streak Viruses

Samples of maize leaves naturally infected with maize streak virus (MSV) from Malawi and South Africa, as well as sugarcane leaves naturally infected with sugarcane streak virus (SSV) from Egypt, were examined by light (LM) and transmission electron, microscopy (TEM). Intranuclear inclusions, detectable by both methods, were found mainly in mesophyll and bundle sheath cells, and less frequently in vascular parenchyma and immature phloem cells. At higher TEM magnifications, these inclusions consisted, of crystalline or noncrystalline aggregates of isometric geminivirus–like particles (VLP) that occurred either singly or in geminate arrays. Cytopathological changes in these cells were confined to the nuclei, which were usually larger than normal, with peripheral chromatin and nucleoli. The nuclear envelope of some inclusion–containing nuclei was ruptured, and occasionally a crystal of VLP was found in the cytoplasm of cells in which no intact nuclei were detected. No differences in cytopathology were found between MSV and SSV, or between the two MSV isolates examined.     

Genetic gain and cost efficiency of marker-assisted selection of maize for improved resistance to multiple foliar pathogens

Northern corn leaf blight (NCLB) caused by Exserohilum turcicum, gray leaf spot (GLS) caused by Cercospora zeae-maydis and maize streak caused by maize streak Mastrevirus (MSV) are the most destructive foliar diseases limiting maize production in sub-Saharan Africa. Most foliar diseases of maize are managed using quantitative (partial) resistance, and previous studies have reported quantitative trait loci associated with host resistance (rQTL). Our objective was to compare the genetic gain and costs resulting from phenotypic, genotypic, and marker-assisted selection of partially inbred lines derived from many families for resistance to infection by three foliar pathogens. We developed a population of 410 F2:3 families by crossing inbred line CML202 with a breeding line designated VP31. These families were planted in nurseries inoculated separately with each pathogen. We conducted one cycle of early generation pedigree selection using three different procedures, phenotypic, genotypic, and marker/phenotypic index, for improvement of resistance to each pathogen. We used simple sequence repeat (SSR) markers flanking six target rQTL associated with partial resistance. Broad- and narrow-sense heritability estimates were also obtained for the F2:3 families, and selected and non-selected F2:4 families. Genetic gains resulting from the selection procedures were determined. Gene action of the candidate rQTL was determined using orthogonal contrasts. Estimates of costs based on lower boundary values indicated that the cost of marker-based selection was lower than that of phenotypic selection. Our results indicate that molecular markers linked to target rQTL can facilitate pyramiding resistance to multiple diseases during early generation pedigree selection.     

The binding motifs for Ac transposase are absolutely required for excision of Ds1 in maize

A reverse genetic system for studying excision of the transposable elementDs1 in maize plants has been established previously. In this system, theDs1 element, as part of the genome of maize streak virus (MSV), is introduced into maize plants via agroinfection. In the presence of theAc element, excision ofDs1 from the MSV genome results in the appearance of viral symptoms on the maize plants. Here, we used this system to study DNA sequences requiredin cis for excision ofDs1. TheDs1 element contains theAc transposase binding motif AAACGG in only one of its subterminal regions (defined here as the 5′ subterminal region). We showed that mutation of these motifs abolished completely the excision capacity ofDs1. This is the first direct demonstration that the transposase binding motifs are essential for excision. Mutagenesis with oligonucleotide insertions in the other (3′) subterminal region resulted in elements with either a reduced or an increased excision efficiency, indicating that this subterminal region also has an important function.     

Mechanism of Ds1 excision from the genome of maize streak virus

Summary We have previously shown that the maize transposable element Ds1 introduced into maize plants by agroinfection can be excised from the genome of geminivirus maize streak virus (MSV). Excision depended strictly on the presence of an active Ac element in the plants. In this study, the excision products or footprints left in the MSV genome after Ds1 excision were extensively characterized and the effects of flanking sequences on Ds1 excision were analysed. Most types of footprints obtained were comparable to those described for Ds1 excision in the maize genome, and could be explained by the models proposed for excision of plant transposable elements. In two revertants, however, some terminal sequences of the Ds1 element were found to have been left behind at the excision site. The finding of this novel type of Ds1 footprint indicated that gene conversion events occurred during and/or after Ds1 excision from the MSV genome. A partial deletion of one copy of the 8 by duplications flanking the Ds1 element had no effect on the frequency or on the types of footprints of Ds1 excision from the MSV genome. Thus, the duplicated 8 by sequences flanking the transposable element are not involved in Ds1 excision. These results, as well as a statistical analysis of the modifications of the bases flanking the Ds1 element after excision, are discussed in terms of excision models.     

Maize streak virus-resistant transgenic maize: a first for Africa

In this article, we report transgene-derived resistance in maize to the severe pathogen maize streak virus (MSV). The mutated MSV replication-associated protein gene that was used to transform maize showed stable expression to the fourth generation. Transgenic T₂ and T₃ plants displayed a significant delay in symptom development, a decrease in symptom severity and higher survival rates than non-transgenic plants after MSV challenge, as did a transgenic hybrid made by crossing T₂ Hi-II with the widely grown, commercial, highly MSV-susceptible, white maize genotype WM3. To the best of our knowledge, this is the first maize to be developed with transgenic MSV resistance and the first all-African-produced genetically modified crop plant.     

Maize streak virus genes essential for systemic spread and symptom development

The entire genome of single component geminiviruses such as maize streak virus (MSV) consists of a single-stranded circular DNA of ~2.7 kb. Although this size is sufficient to encode only three average sized proteins, the virus is capable of causing severe disease of many monocots with symptoms of chlorosis and stunting. We have identified viral gene functions essential for systemic spread and symptom development during MSV infection. Deletions and gene replacement mutants were created by site-directed mutagenesis and insertion between flanking MSV or reporter gene sequences contained in Agrobacterium T-DNA derived vectors. Following Agrobacterium-mediated inoculation of maize seedlings, the mutated MSV DNAs were excised from these binary vectors by homologous recombination within the flanking sequences. Our analyses show that the capsid gene of MSV, while not required for replication, is essential for systemic spread and subsequent disease development. The `+' strand open reading frame (ORF) located immediately upstream from the capsid ORF and predicted to encode a 10.9 kd protein was also found to be dispensable for replication but essential for systemic spread. By this analysis, MSV sequences that support autonomous replication were localized to a 1.7 kb segment containing the two viral intergenic regions and two overlapping complementary `-' strand ORFs. Despite the inability of the gene replacement mutants to spread systemically, both inoculated and newly developed leaves displayed chlorotic patterns similar to the phenotype observed in certain developmental mutants of maize. The similarity of the MSV mutant phenotype to these developmental mutants is discussed.     

Purification of maize streak virus and its relationship to viruses associated with streak diseases of sugar cane and Panicum maximum

Maize streak virus (MSV) was purified by homogenising infected leaf tissue in 0·01 m pH 3·9 phosphate buffer and clarifying the extract with n-butanol (7 ml/100 ml extract). Purified preparations contained particles 20 nm in diameter, some occurring singly, but most occurring in pairs, forming structures of 30 × 20 nm. The sedimentation coefficients of single and paired particles were 54 and 76 S respectively. When centrifuged in sucrose density gradients preparations made by extracting leaves at pH 3·9 gave a single intense light-scattering zone containing paired particles. Preparations made at pH 5·9 or 7·9 gave one or two additional upper zones containing single particles and fragmented material. Preparations treated with 0·05 or 0·1 m ethylene diamine tetra-acetic acid, disodium salt, (EDTA) contained no paired particles, few single particles and much fragmented material. In immunoelectrophoresis, the major component in preparations without EDTA migrated to the cathode whereas that in EDTA-treated preparations migrated to the anode. Virus isolates from streak-diseased sugarcane and guinea grass (Panicum maximum) were serologically related to MSV and had similar particles with identical sedimentation coefficients. No such particles were seen in purified preparations of healthy maize, sugarcane, or guinea grass. The viruses from sugarcane and guinea grass are probably host-adapted and are referred to correctly as the sugarcane and guinea grass strains of MSV. MSV probably contains single-stranded RNA, and the cryptogram is (R)/1:*/*:S/S:S/Au.