Resistance gene analogs involved in tolerant cassava--geminivirus interaction that shows a recovery phenotype.

The current literature describes recovery from virus-induced symptoms as a RNA silencing defense, but immunity-related genes, including the structurally specific resistance gene analogs (RGAs) that may play a key role in tolerance and recovery is not yet reported. In this study, the transcriptome data of tolerant cassava TME3 (which exhibits a recovery phenotype) and susceptible cassava T200 infected with South African cassava mosaic virus were explored for RGAs. Putative resistance protein analogs (RPAs) with amide-like indole-3-acetic acid-Ile-Leu-Arg (IAA-ILR) and leucine-rich repeat (LRR)-kinase conserved domains were unique to TME3. Common responsive RPAs in TME3 and T200 were the dirigent-like protein, coil-coil nucleotide-binding site (NBS) and toll-interleukin-resistance, disease resistance zinc finger chromosome condensation-like protein (DZC), and NBS-apoptosis repressor with caspase recruitment (ARC)-LRR domains. Mutations in RPAs in the MHD motif of the NBS-ARC2 subdomain associated with the recovery phase in TME3 were observed. Additionally, a cohort of 25 RGAs mined solely during the recovery process in TME3 was identified. Phylogenetic and expression analyses support that diverse RGAs are differentially expressed during tolerance and recovery. This study reveals that in cassava, a perennial crop, RGAs participate in tolerance and differentially accumulate during recovery as a complementary defense mechanism to natural occurring RNA silencing to impair viral replication.

Overexpression of a GmCnx1 gene enhanced activity of nitrate reductase and aldehyde oxidase, and boosted mosaic virus resistance in soybean.

Molybdenum cofactor (Moco) is required for the activities of Moco-dependant enzymes. Cofactor for nitrate reductase and xanthine dehydrogenase (Cnx1) is known to be involved in the biosynthesis of Moco in plants. In this work, a soybean (Glycine max L.) Cnx1 gene (GmCnx1) was transferred into soybean using Agrobacterium tumefaciens-mediated transformation method. Twenty seven positive transgenic soybean plants were identified by coating leaves with phosphinothricin, bar protein quick dip stick and PCR analysis. Moreover, Southern blot analysis was carried out to confirm the insertion of GmCnx1 gene. Furthermore, expression of GmCnx1 gene in leaf and root of all transgenic lines increased 1.04-2.12 and 1.55-3.89 folds, respectively, as compared to wild type with GmCnx1 gene and in line 10 , 22 showing the highest expression. The activities of Moco-related enzymes viz nitrate reductase (NR) and aldehydeoxidase (AO) of T1 generation plants revealed that the best line among the GmCnx1 transgenic plants accumulated 4.25 µg g(-1) h(-1) and 30 pmol L(-1), respectively (approximately 2.6-fold and 3.9-fold higher than non-transgenic control plants).In addition, overexpression ofGmCnx1boosted the resistance to various strains of soybean mosaic virus (SMV). DAS-ELISA analysis further revealed that infection rate of GmCnx1 transgenic plants were generally lower than those of non-transgenic plants among two different virus strains tested. Taken together, this study showed that overexpression of a GmCnx1 gene enhanced NR and AO activities and SMV resistance, suggesting its important role in soybean genetic improvement.

Trypsin inhibitors from Capsicum baccatum var. pendulum leaves involved in Pepper yellow mosaic virus resistance.

Several plant organs contain proteinase inhibitors, which are produced during normal plant development or are induced upon pathogen attack to suppress the enzymatic activity of phytopathogenic microorganisms. In this study, we examined the presence of proteinase inhibitors, specifically trypsin inhibitors, in the leaf extract of Capsicum baccatum var. pendulum inoculated with PepYMV (Pepper yellow mosaic virus). Leaf extract from plants with the accession number UENF 1624, which is resistant to PepYMV, was collected at 7 different times (0, 24, 48, 72, 96, 120, and 144 h). Seedlings inoculated with PepYMV and control seedlings were grown in a growth chamber. Protein extract from leaf samples was partially purified by reversed-phase chromatography using a C2/C18 column. Residual trypsin activity was assayed to detect inhibitors followed by Tricine-SDS-PAGE analysis to determine the N-terminal peptide sequence. Based on trypsin inhibitor assays, trypsin inhibitors are likely constitutively synthesized in C. baccatum var. pendulum leaf tissue. These inhibitors are likely a defense mechanism for the C. baccatum var. pendulum- PepYMV pathosystem.

Development of an Efficient Virus Induced Gene Silencing Strategy in the Non-Model Wild Ginger-Zingiber zerumbet and Investigation of Associated Proteome Changes.

Zingiber zerumbet (Zingiberaceae) is a wild, tropical medicinal herb that shows a high degree of resistance to diseases affecting cultivated ginger. Barley stripe mosaic virus (BSMV) silencing vectors containing an endogenous phytoene desaturase (PDS) gene fragment were agroinfiltrated into young leaves of Z. zerumbet under controlled growth conditions to effect virus-induced gene silencing (VIGS). Infiltrated leaves as well as newly emerged leaves and tillers showed visual signs of PDS silencing after 30 days. Replication and systemic movement of the viral vectors in silenced plants were confirmed by RT-PCR. Real-time quantitative PCR analysis verified significant down-regulation of PDS transcripts in the silenced tissues. Label-free proteomic analysis was conducted in leaves with established PDS transcript down regulation and buffer-infiltrated (mock) leaves. A total of 474 proteins were obtained, which were up-regulated, down-regulated or modulated de novo during VIGS. Most of these proteins were localized to the chloroplast, as revealed by UniprotKB analysis, and among the up-regulated proteins there were abiotic stress responsive, photosynthetic, metabolic and membrane proteins. Moreover, the demonstration of viral proteins together with host proteins proved successful viral infection. We report for the first time the establishment of a high-throughput gene functional analysis platform using BSMV-mediated VIGS in Z. zerumbet, as well as proteomic changes associated with VIGS.

Complementation of the host range restriction of southern cowpea mosaic virus in bean by southern bean mosaic virus.

Vigna unguiculata (cowpea) and Phaseolus vulgaris (common bean) are permissive hosts for southern cowpea mosaic virus (SCPMV) and southern bean mosaic virus (SBMV), respectively. Neither of these two sobemoviruses systemically infects the permissive host of the other. Although bean cells are permissive for SCPMV RNA synthesis, they do not support the assembly of this virus. Thus, the host range restriction of SCPMV in bean may occur at the level of movement and may involve the inability of SCPMV to assemble in this host. In this study, it was demonstrated that SCPMV accumulates in an encapsidated form in the inoculated and systemic leaves of bean plants following coinoculation with SBMV. No evidence was observed that the SCPMV that accumulated in coinoculated bean plants had an altered host range relative to wild-type SCPMV. These results suggested that SBMV complemented the host range restriction of SCPMV in bean. Additional experiments demonstrated that cowpea protoplasts are permissive for SBMV RNA synthesis and assembly. It was concluded from these results that the host range restriction of SBMV in cowpea occurs at the level of movement. In mixed infections of cowpea with SCPMV and SBMV, the latter was recovered from the inoculated but not the systemic leaves. Its recovery from the inoculated leaves, however, was not dependent on the presence of SCPMV in the inoculum. From these results, it was concluded that SCPMV did not complement the host range restriction of SBMV in cowpea.

Identification of viral genes required for cell-to-cell movement of southern bean mosaic virus.

Inoculation of Vigna unguiculata (cowpea) with transcripts synthesized in vitro from a genome-length cDNA clone of the cowpea strain of southern bean mosaic virus (SBMV-C) resulted in a systemic SBMV-C infection of this host. Capped RNA was about five times more infectious than uncapped RNA as determined by a local lesion assay. The SBMV-C cDNA clone was also used for mutagenesis of the four SBMV-C open reading frames (ORFs). ORF1, ORF3, and coat protein (CP) mutants were not infectious in cowpea. Electroporation of cowpea protoplasts with mutant transcripts demonstrated that the ORF1, ORF3, and CP gene products were not required for SBMV-C RNA synthesis, and the ORF1 and ORF3 gene products were not required for SBMV-C assembly. From these results, it was concluded that the ORF1 and ORF3 proteins and the CP are required for SBMV-C cell-to-cell movement. One of the ORF3 mutants pSBMV2-UAA1833 contained a nonsense codon between the predicted -1 ribosomal frameshift site (SBMV-C nucleotides 1796-1802) and a potential ORF3 translation initiation codon at SBMV-C nucleotide 1895. The lack of infectivity of this mutant suggested that ORF3 was expressed by a -1 ribosomal frameshift in ORF2 rather than by initiation of translation at nucleotide 1895.

Complementation of a potato virus X mutant mediated by bombardment of plant tissues with cloned viral movement protein genes.

Microprojectile bombardment was used to examine the transport function of the 25 kDa movement protein (MP) encoded in the triple gene block of potato virus X (PVX). A 25 kDa MP-defective full-length cloned PVX genome carrying a beta-glucuronidase (GUS) reporter gene was co-bombarded with 35S promoter constructs containing either the 25 kDa MP gene of wild-type PVX, the MP gene of either of two tobamoviruses (tomato mosaic virus or crucifer tobamovirus), red clover necrotic mosaic dianthovirus (RCNMV) or brome mosaic bromovirus (BMV). When inoculated alone, the MP-defective PVX was unable to move out of the inoculated cell, as visualized by in situ staining for GUS activity. However, cell-to-cell movement of the mutant PVX genome was restored by co-inoculation with 35S constructs containing the MP cDNA of PVX, either tobamovirus or RCNMV. The BMV MP construct did not complement movement of the defective PVX. These results show that co-bombardment of cDNA of an MP-defective virus with plasmids designed to express MP of other viruses could be used as a fast and simple method for transcomplementation experiments.

Host-controlled cell-to-cell movement of a hybrid barley stripe mosaic virus expressing a dianthovirus movement protein.

The triple gene block (TGB) of barley stripe mosaic virus (BSMV), coding for viral movement proteins (MPs), was replaced by the single MP gene of red clover necrotic mosaic virus (RCNMV). Accumulation of the hybrid virus in barley plants (the selective host for BSMV) was reduced compared to BSMV. The hybrid virus induced small necrotic local lesions on Chenopodium amaranticolor leaves and did not infect Nicotiana clevelandii (the selective host for RCNMV). The hybrid virus accumulated in the inoculated leaves of Nicotiana benthamiana, but not in the upper noninoculated leaves. Thus the RCNMV MP gene substituted for the BSMV TGB in cell-to-cell movement, but not in systemic spread. Hybrid virus movement was efficient only in N. benthamiana, the common host for BSMV and RCNMV. These data point to the involvement of host-specific factors in the function of virus-coded transport determinants.

Structure of Sesbania mosaic virus at 3 A resolution.

Sesbania mosaic virus (SMV) is an isometric, ss-RNA plant virus found infecting Sesbania grandiflora plants in fields near Tirupathi, South India. The virus particles, which sediment at 116 S at pH 5.5, swell upon treatment with EDTA at pH 7.5 resulting in the reduction of the sedimentation coefficient to 108 S. SMV coat protein amino acid sequence was determined and found to have approximately 60% amino acid sequence identity with that of southern bean mosaic virus (SBMV). The amino terminal 60 residue segment, which contains a number of positively charged residues, is less well conserved between SMV and SBMV when compared to the rest of the sequence. The 3D structure of SMV was determined at 3.0 A resolution by molecular replacement techniques using SBMV structure as the initial phasing model. The icosahedral asymmetric unit was found to contain four calcium ions occurring in inter subunit interfaces and three protein subunits, designated A, B and C. The conformation of the C subunit appears to be different from those of A and B in several segments of the polypeptide. These observations coupled with structural studies on SMV partially depleted of calcium suggest a plausible mechanism for the initiation of the disassembly of the virus capsid.

Improved diagnosis of cowpea aphid-borne mosaic virus in Africa: significance for cowpea seed-indexing, breeding programs and potyvirus taxonomy.

Large-scale surveys in Africa for blackeye cowpea mosaic (B1CMV) and cowpea aphid-borne mosaic (CABMV) showed that several CABMV isolates from Southern Africa were either not or poorly recognized by monoclonal antibodies prepared to isolates collected in West Africa. Selection of three new monoclonal antibodies prepared against the Maputo (Mozambique) isolate of CABMV, and their incorporation into a revised panel of monoclonal antibodies, resulted in the assignment of four of these new CABMV isolates to existing serotypes (II, IV, and V) and three others to a new serotype (VI). The South African isolate of passiflora mosaic virus was shown to be related to CABMV isolates in serotype IV. It is proposed that CABMV isolates be assembled into a distinct species in the legume-infecting, aphid-transmissible potyviruses.

Virion formation is required for the long-distance movement of red clover necrotic mosaic virus in movement protein transgenic plants.

The red clover necrotic mosaic dianthovirus (RCNMV) genome is split between two single-stranded RNA species. The polycistronic RNA-1 encodes the viral RNA polymerase and capsid protein (CP) and the monocistronic RNA-2 encodes the 35 kDa cell-to-cell movement protein (MP). Nicotiana benthamiana plants transformed with the RCNMV MP gene were generated. When inoculated onto the MP transgenic plants, cell-to-cell movement of RNA-1 occurred at a rate similar to wild-type virus. However, long-distance (leaf-to-leaf) movement of RNA-1 was not observed. Neither CP nor virions were detected in the inoculated leaves of the MP transgenic plants. When RNA-1 was coinoculated with RNA-2 mutants, which do not express a functional MP, onto MP transgenic plants, CP and virions were readily detected and a systemic infection resulted. These results demonstrate that both RNA-1 and RNA-2 are necessary for the accumulation of both CP and virions. Furthermore, CP accumulation was found to be required for long-distance movement of RCNMV. Therefore, these data provide evidence that CP, in the form of virions, is necessary for the long-distance movement of RCNMV.

Failure of long-distance movement of southern bean mosaic virus in a resistant host is correlated with lack of normal virion formation.

Sunn-hemp mosaic tobamovirus (SHMV) facilitated the spread of the cowpea strain of southern bean sobemovirus (SBMV-C) only in inoculated leaves of common bean (Phaseolus vulgaris L. cv. Bountiful), a resistant host for SBMV-C. Tissue prints of bean primary leaves doubly inoculated with SHMV and SBMV-C, developed by Western blotting, showed the presence of the SBMV-C capsid antigen in the mesophyll and epidermis, but no antigen was detected in the conducting bundles. Typical SBMV-C virions were not seen in electron micrographs of immunogold-labelled mesophyll cells; instead, specifically labelled, amorphous protein clumps were found in the vacuole. Particles of smaller diameter than that of typical SBMV-C virions were specifically trapped by SBMV antibodies following immunosorbent electron microscopy of extracts from doubly infected leaves. SBMV-C coat protein from infected Vigna unguiculata L. (cowpea) and bean plants showed no difference in its mobility following electrophoresis in denaturing SDS-polyacrylamide gels. Lack of efficient assembly of SBMV-C virions does not impede cell-to-cell movement of the virus in doubly infected leaves of bean, yet it is probably an important factor in determining the inability of SBMV-C to move into and/or through the vascular system of this host.

The involvement of cowpea mosaic virus M RNA-encoded proteins in tubule formation.

On the surface of cowpea protoplasts inoculated with cowpea mosaic virus (CPMV), tubular structures containing virus particles have been found. Such tubular structures are thought to be involved in cell-to-cell movement of CPMV in cowpea plants. To study the involvement of the 58K/48K and capsid proteins of CPMV in the formation of the tubular structures, mutations were introduced into M cDNA clones from which infectious transcripts could be derived. No tubules were found on protoplasts inoculated with a mutant that fails to produce the 48K protein nor with a mutant that has a deletion in the 48K coding region, suggesting that the 48K protein is essential for this process. However, a possible role of the 58K protein in tubule formation could not be excluded. A mutant that fails to produce the capsid proteins did produce tubules and therefore the capsid proteins are not involved in the formation of the tubular structures. Electron microscopic analysis revealed that the tubules produced by this mutant are, apart from the absence of virus particles, morphologically identical to the tubules formed by the wild-type virus.

Signal for potyvirus-dependent aphid transmission of potato aucuba mosaic virus and the effect of its transfer to potato virus X.

A British isolate of potato aucuba mosaic potexvirus (PAMV) was transmitted by aphids (Myzus persicae) which had fed previously on a source of potato Y potyvirus (PVY). Nucleotide sequence analysis of the PAMV coat protein gene indicated that amino acid residues 14 to 16 from the N terminus of the coat protein have the sequence DAG, which is also found in the coat proteins of potyviruses and is required for their aphid transmissibility. A recombinant virus isolate (TXPA7) was produced in which a segment of the coat protein gene of PAMV encoding the 40 N-terminal amino acids was inserted in the genome of potato X potexvirus (PVX) in place of the segment encoding the 28 N-terminal amino acids of PVX coat protein. This isolate, and a second similar recombinant (TXPA5) in which the DAG motif was changed to YTS, were mechanically transmissible to intact plants, in which they caused slightly milder symptoms than PVX. Particles of TXPA7 reacted in immunosorbent electron microscopy with PVX- and PAMV-specific antibodies and so were antigenically distinguishable from PAMV and PVX particles, which reacted only with their homologous antibody, and from TXPA5 particles, which reacted only with the PVX antibody. Recombinant TXPA7 was transmitted by aphids that had already fed on a source of PVY whereas TXPA5 and PVX were not. TXPA7 was not transmitted by aphids that had not fed on a PVY source. It is concluded that (i) the potyvirus-dependent aphid transmissibility of PAMV results from possession of a domain which includes the DAG motif and is located near the N terminus of the virus coat protein, and (ii) potyvirus-dependent aphid transmissibility can be conferred on PVX, a non-aphid-borne potexvirus, by substituting this domain for the N-terminal part of its coat protein.

The 5' untranslated region from pea seedborne mosaic potyvirus RNA as a translational enhancer in pea and tobacco protoplasts.

We have exploited the transient expression of foreign genes introduced into plant protoplasts to investigate the effect of the pea seedborne mosaic potyvirus (PSbMV) 5' untranslated region (5'UTR) on the level of gene expression in pea and tobacco protoplasts. The plant viral 5'UTRs were found to increase translation significantly in comparison to a plasmid containing no 5'UTR of viral origin. The enhancement effect of the 5'UTRs of PSbMV and tobacco etch potyvirus (TEV) was found to be similar in pea and tobacco protoplasts, indicating a host-independent role of the potyviral 5'UTRs in enhancing gene expression. Translational enhancement of the two potyviral 5'UTRs was similar to that of the 5'UTR of tobacco mosaic virus (TMV). This observation makes it attractive to use potyviral 5'UTRs as general translational enhancers in future genetic transformations of plants.

Improvements of the infectivity of in vitro transcripts from cloned cowpea mosaic virus cDNA: impact of terminal nucleotide sequences.

Full-length DNA copies of both B- and M-RNA of cowpea mosaic virus (CPMV) were constructed downstream from a T7 promoter. By removal of nucleotides from the promoter sequence, B- and M-RNA-like transcripts with varying numbers of additional nonviral sequences at the 5' end were obtained upon transcription with T7 RNA polymerase. The infectivity of the transcripts in cowpea protoplasts was greatly affected by only a few extra nonviral nucleotides at the 5' end. The addition of about 400 nonviral nucleotides at the 3' end did not have any effect. Using the most infectious transcripts, in 40% of the cowpea protoplasts replication and expression of B-RNA like transcripts were observed and in 10% of the protoplasts both B- and M-RNA-like transcripts multiplied. Moreover, cowpea plants could also be infected with these transcripts. Sequence analysis showed that the 5' terminus of the M-RNA transcripts and the 3' terminus of the B-RNA transcripts were completely restored during replication in plants, including a poly(A) tail of variable length. Swapping experiments have been used to identify an influential point mutation in the coding region for the viral polymerase of a noninfectious B transcript. This experiment demonstrates the potential of the optimized infection system for future analysis of virus-encoded functions.

Analysis of sequences involved in cowpea mosaic virus RNA replication using site-specific mutants.

Using a full-length cDNA clone of cowpea mosaic virus (CPMV) B-RNA from which infectious transcripts can be generated, we examined the influence of a sequence of 11 nucleotides, UUUUAUUAAAA, comprising the nucleotides 5883 to 5893 in the 3' noncoding region of B-RNA, on viral RNA replication. This sequence is not only present in B-RNA but also in M-RNA and represents the 7 nucleotides preceding the poly(A) tail and the first four A residues of the poly(A) tail. Replication of B-RNA transcripts derived from a series of mutants in this region was tested in cowpea plants and protoplasts. Only mutant transcripts with minor modifications appeared able to replicate, which indicates that the region has a function in viral RNA replication. In addition, the results suggest the existence of a hairpin loop in this region. Those transcripts with deletions which disturb the putative hairpin structure have decreased specific infectivities. Mutant transcripts reversed stepwise to the wild-type sequence during replication in plants. This observation strengthens the idea that the sequence of 11 nucleotides has a function in viral RNA replication.

Infectious in vitro transcripts from cowpea chlorotic mottle virus cDNA clones and exchange of individual RNA components with brome mosaic virus.

Complete cDNA copies of genomic RNA1, RNA2, and RNA3 of cowpea chlorotic mottle virus (CCMV) were cloned 1 base downstream from a T7 RNA polymerase promoter. The mixture of capped in vitro transcripts from all three clones produced normal CCMV infections in barley protoplasts and cowpea plants. By using transcripts from these clones and from a similar set of biologically active clones of the related brome mosaic virus (BMV), all possible single component exchanges between the BMV and CCMV tripartite genomes were tested. Viral RNA replication was not observed with any heterologous combination of RNA1 and RNA2, which encode trans-acting viral RNA replication factors. However, substitution of the heterologous RNA3 into either genome produced viable hybrid viruses, both of which replicated in barley protoplasts and produced lesions on Chenopodium hybridum, a local lesion host for both parent viruses. In hybrid infections, BMV and CCMV coat proteins each readily packaged RNAs from the heterologous virus, but BMV RNAs were replicated to a higher level than CCMV RNAs, even when trans-acting RNA replication factors were provided by CCMV genes. Neither hybrid systemically infected the natural host of either parent virus, suggesting that host specificity determinants in BMV and CCMV are encoded by RNA3 and at least one other genomic RNA.

Binding of cowpea chlorotic mottle virus to cowpea protoplasts and relation of binding to virus entry and infection.

Cowpea chlorotic mottle virus (CCMV) and cowpea protoplasts were used to study initial interactions between virus and protoplast. Protoplasts and virus were incubated under varying conditions of temperature, pH, ionic strength, and the presence of added compounds. Both the amount of 35S-labeled virus bound to protoplasts and the percentage of infected cells were determined. At 0 and 25 degrees the amount of virus associated with protoplasts increased with the amount of virus added. With inoculum of 25 x 10(6) virus particles per protoplast, 4 x 10(3) and 14 x 10(3) particles per protoplast were bound at 0 and 25 degrees, respectively. In the presence of polyethylene glycol, 85 x 10(3) associated particles per protoplast were bound at both temperatures and ca. 50% of the protoplasts became infected. No infection occurred in the absence of PEG. Variation of pH or ionic strength in the absence of PEG caused little to no change in binding and no infection. In the presence of PEG, increase of pH resulted in lower binding, but infectivity was not affected. Increasing ionic strength, however, increased both binding and infectivity. The presence of unlabeled CCMV, tobacco mosaic virus coat protein, bovine serum albumin, and polycations during inoculation in the absence of PEG decreased the amount of bound CCMV. In contrast, CCMV coat protein, which has a positively charged N-terminal arm, increased binding. In the presence of PEG the effects were similar, although larger amounts of virus were bound. The percentage of infection was reduced by all additives to 5-25%. Addition of ammonium chloride, which inhibits endocytotic virus uptake in animal cells, during inoculation as well as in culture media, did not reduce infectivity. These data do not support a specific receptor-mediated endocytotic uptake of virus but favor a nonspecific mechanism of entry, possibly through membrane lesions. Observations in the electron microscope support the latter mechanism.

Specificity of the actinomycin-D-sensitive function of some RNA plant viruses.

The actinomycin-D(AMD)-sensitive step of replication of three plant viruses - cowpea chlorotic mottle virus (CCMV), cowpea mosaic virus, and the cowpea strain of tobacco mosaic virus - in cowpea was specific for each virus. Different combinations of the viruses were examined in which leaves were inoculated with one virus and incubated for 48 h until multiplication was almost resistant to AMD. At that time, the same leaves were inoculated with a different virus and the ability of AMD to inhibit multiplication of the second virus was determined at intervals afterwards. In all combinations tested, the first virus had no effect on the sensitivity of the second virus to AMD. Also, resistance to AMD, which appeared to spread from cell to cell in advance of the virus in mechanically inoculated leaves, did not spread appreciably faster than CCMV into systemically infected upper leaves.