First Report of Maize chlorotic mottle virus and Maize Lethal Necrosis in Kenya.

In September 2011, a high incidence of a new maize (Zea mays L.) disease was reported at lower elevations (1,900 m asl) in the Longisa division of Bomet County, Southern Rift Valley, Kenya. The disease later spread to the Narok South and North and Naivasha Districts. By March 2012, the disease was reported at up to 2,100 m asl. Diseased plants had symptoms characteristic of virus diseases: a chlorotic mottle on leaves, developing from the base of young whorl leaves upward to the leaf tips; mild to severe leaf mottling; and necrosis developing from leaf margins to the mid-rib. Necrosis of young leaves led to a "dead heart" symptom, and plant death. Severely affected plants had small cobs with little or no grain set. Plants frequently died before tasseling. All maize varieties grown in the affected areas had similar symptoms. In these regions, maize is grown continuously throughout the year, with the main planting season starting in November. Maize streak virus was present, but incidence was low (data not shown). Infected plants were distributed throughout affected fields, with heavier infection along field edges. High thrips (Frankliniella williamsi Hood) populations were present in sampled fields, but populations of other potential disease vectors, such as aphids and leafhoppers, were low. Because of the high thrips populations and foliar symptoms, symptomatic plants were tested for the presence of Maize chlorotic mottle virus (MCMV) (3) using tissue blot immunoassay (TBIA) (1). Of 17 symptomatic leaf samples from each Bomet and Naivasha, nine from Bomet and all 17 from Naivasha were positive for MCMV. However, the observed symptoms were more severe than commonly associated with MCMV, suggesting the presence of maize lethal necrosis (MLN), a disease that results from maize infection with both MCMV and a potyvirus (4). Therefore, samples were tested for the presence of Sugarcane mosaic virus (SCMV), which is present in Kenya (2). Twenty-seven samples were positive for SCMV by TBIA, and 23 of 34 samples were infected with both viruses. Virus identities were verified with reverse-transcription (RT)-PCR (Access RT-PCR, Promega) and MCMV or SCMV-specific primers. MCMV primers (2681F: 5'-ATGAGAGCAGTTGGGGAATGCG and 3226R: 5'-CGAATCTACACACACACACTCCAGC) amplified the expected 550-bp product from three leaf samples. Amplicon sequences were identical, and had 95 to 98% identity with MCMV sequences in GenBank. SCMV primers (8679F: 5'-GCAATGTCGAAGAAAATGCG) and 9595R: 5'-GTCTCTCACCAAGAGACTCGCAGC) amplified the expected 900-bp product from four leaf samples. Amplicon sequences had 96 to 98% identity, and were 88 to 96% identical with SCMV sequences in GenBank. To our knowledge, this is the first report of MCMV and of maize coinfection with MCMV and SCMV associated with MLN in Kenya and Africa. MLN is a serious threat to farmers in the affected areas, who are experiencing extensive to complete crop loss. References: (1) P. G. S. Chang et al. J. Virol. Meth. 171:345, 2011. (2) Delgadillo Sanchez et al. Rev. Mex. Fitopat. 5:21, 1987. (3) Jiang et al., Crop Prot. 11:248, 1992. (4) R. Louie, Plant Dis. 64:944, 1980.

Maize chlorotic mottle machlomovirus expresses its coat protein from a 1.47-kb subgenomic RNA and makes a 0.34-kb subgenomic RNA.

Analysis of double-stranded RNAs produced in maize plants infected with maize chlorotic mottle machlomovirus (MCMV) and Northern blots of total RNA from infected plants or protoplasts showed two subgenomic RNAs (sgRNAs). Primer extension was used to map these sgRNAs, which are 1.47 and 0.34 kb long. The transcription start sites are nucleotide (nt) 2970 or 2971 for sgRNA1 and nt 4101 for sgRNA2. The 5' ends of the sgRNAs are similar to one another and to the 5' end of genomic RNA, and 11 nt sequences immediately upstream of their transcription start sites are similar. The location of the sgRNA1 transcription start site indicates that MCMV expresses a 7-kDa open reading frame (ORF) from nt 2995 to 3202 instead of the predicted 9-kDa ORF from nt 2959 to 3202. In protoplast inoculation experiments, a silent mutation at nt 2965 and a 4-nt change at nt 2959-2962 stopped the synthesis of sgRNA1 and expression of the coat protein ORF, which begins more than 400 nt downstream. Replication of MCMV does not require the expression of any of the ORFs encoded on sgRNA1. SgRNA2 has the potential to encode 2.3-, 2.7-, and 4. 6-kDa peptides, but the function, if any, of sgRNA2 is unknown.

Maize chlorotic mottle machlomovirus and wheat streak mosaic rymovirus concentrations increase in the synergistic disease corn lethal necrosis.

Corn lethal necrosis (CLN) is caused by the synergistic interaction between maize chlorotic mottle machlomovirus (MCMV) and any potyvirus which infects cereals. Interactions between MCMV and wheat streak mosaic rymovirus (WSMV) in N28Ht corn produced MCMV concentrations that averaged 3.3- to 11.2-fold higher in doubly infected plants than the average concentrations in plants inoculated with MCMV. MCMV-negative sense RNA concentrations were similarly increased, and the ratio of full-length to subgenomic RNA was the same in singly and doubly infected plants. Contrary to most synergisms involving a potyvirus, WSMV infections were enhanced by the presence of MCMV. WSMV infection rates were higher when plants were coinoculated with MCMV, and the difference in infection rates was more pronounced at higher temperatures. Under conditions favorable for establishing high WSMV infection rates (cooler temperatures and high light intensity), WSMV concentrations in doubly infected plants averaged 2.1- to 3.1-fold higher than those in singly inoculated plants. Doubly inoculated plants with the lowest WSMV levels also had the lowest MCMV concentrations, but the concentrations of MCMV and WSMV in the most heavily infected plants did not directly correlate. These results suggest that there are genes in both MCMV and WSMV which directly or indirectly affect the replication and/or spread of the other virus in CLN.

Transcripts of a maize chlorotic mottle virus cDNA clone replicate in maize protoplasts and infect maize plants.

A full-length cDNA clone (pMCM41) was constructed to contain the exact 5' end of MCMV behind a T7 RNA polymerase promoter and a Smal site at the 3' end. Uncapped RNA synthesized from pMCM41 has the exact 3' end of viral RNA (vRNA) but is missing the cap found on vRNA. This RNA was infectious in protoplasts from black Mexican sweet (BMS) maize (Zea mays) suspension cultures. Uncapped transcripts were also infectious when inoculated onto maize plants and produced an infection indistinguishable from vRNA-inoculated plants. Capped pMCM41 transcripts which initiated at position +2 of the cDNA clone, as well as capped or uncapped RNA synthesized from a clone containing an extra G between the T7 promoter and the 5' end of MCMV sequence (pMCM721), were less infectious than uncapped pMCM41 transcripts in BMS protoplasts. The transcripts one nucleotide longer or shorter than uncapped pMCM41 transcripts were not able to infect maize plants.

The complete nucleotide sequence of the maize chlorotic mottle virus genome.

The complete nucleotide sequence of the maize chlorotic mottle virus (MCMV) genome has been determined to be 4437 nucleotides. The viral genome has four long open reading frames (ORFs) which could encode polypeptides of 31.6, 50, 8.9 and 25.1 kd. If the termination codons, for the polypeptides encoded by the 50 and 8.9 kd ORFs are suppressed, readthrough products of 111 and 32.7 kd result. The 31.6 and 50 kd ORFs overlap for nearly the entire length of the 31.6 kd ORF. Striking amino acid homology has been observed between two potential polypeptides encoded by MCMV and polypeptides encoded by carnation mottle virus (CarMV) and turnip crinkle virus (TCV). The 25.1 kd ORF most likely encodes the capsid protein. The similar genome organization and amino acid sequence homology of MCMV with CarMV and TCV suggest an evolutionary relationship with these members of the carmovirus group.

Developmentally regulated plant genes: the nucleotide sequence of a wheat gliadin genomic clone.

Gliadins, the major wheat seed storage proteins, are encoded by a multigene family. Northern blot analysis shows that gliadin genes are transcribed in endosperm tissue into two classes of poly(A)+ mRNA, 1400 bases (class I) and 1600 bases (class II) in length. Using poly(A)+ RNA from developing wheat endosperm we constructed a cDNA library from which a number of clones coding for alpha/beta and gamma gliadins were identified by hybrid-selected mRNA translation and DNA sequencing. These cDNA clones were used as probes for the isolation of genomic gliadin clones from a wheat genomic library. One such genomic clone was characterized in detail and its DNA sequence determined. It contains a gene for a 33-kd alpha/beta gliadin protein (a 20 amino acid signal peptide and a 266 amino acid mature protein) which is very rich in glutamine (33.8%) and proline (15.4%). The gene sequence does not contain introns. A typical eukaryotic promoter sequence is present at -104 (relative to the translation initiation codon) and there are two normal polyadenylation signals 77 and 134 bases downstream from the translation termination codon. The coding sequence contains some internal sequence repetition, and is highly homologous to several alpha/beta gliadin cDNA clones. Homology to a gamma-gliadin cDNA clone is low, and there is no homology with known glutenin or zein cDNA sequences.

Perturbation of the mitochondrial lysine tRNA population by virus-induced transformation or stress of mammalian cells: functional properties and nucleotide sequence of a mitochondrially associated lysine tRNA.

Eleven isoaccepting lysine tRNAs from mammalian sources are demonstrable by RPC-5 chromatography and polyacrylamide gel electrophoresis. The appearance and amounts of these isoacceptors varies with the source and growth state of cells. One isoacceptor, tRNALys6, observed in preparations of tRNA from some virus-transformed cells in culture, has been characterized by determining functional properties, cellular location, and its nucleotide sequence. tRNALys6 responds primarily to the lysine codon AAA, but it is not used efficiently in a wheat germ translational system in vitro. Compared with lysine isoacceptors 1, 2, 4, 5a, and 5, [3H]lysine appears in vivo in tRNALys6 with a delay of about 3 h. This delay may in part be a result of a less functional tRNA, but a compartmented state of tRNALys6 also appears to be important. tRNALys6 is associated with mitoplasts prepared from KA31 fibroblasts. The nucleotide sequence of tRNALys6 was determined by rapid postlabeling procedures involving limited hydrolysis in formamide, 32P-labeling of 5' ends of fragments with polynucleotide kinase, separation of the nested set of fragments in polyacrylamide denaturing gels, release of 5'-labeled nucleotides with RNase T2, and identification of the released nucleotides by chromatography on PEI cellulose. Confirmation of the positions of major nucleotides was done by using limited digestions by RNases of tRNALys6 labeled with 32P on the 3' terminus in a gel readout procedure. The nucleotide sequence of tRNALys6 differs from that of cytoplasmic lysine tRNAs and mammalian mitochondrial lysine tRNAs. It contains U*, an unidentified modified uridine occurring in the anticodon of some mitochondrial tRNAs. tRNALys6 appears to occur in very limited amounts, or not at all, in most cells unless stressed, but when present it is associated with mitochondria, although it is probably coded in the nucleus.

Comparison of complexes containing lysyl-tRNA synthetase from normal and virus-transformed cells.

We compared the lysyl-tRNA synthetases from normal (Balb/3T3) and murine sarcoma virus-transformed (KA31) mouse fibroblasts. In agreement with several other reports of mammalian systems, the lysyl-tRNA synthetases from these cells occurred in very large postmicrosomal complexes as determined by gel filtration on agarose columns. Arginyl-, isoleucyl-, methionyl-, phenylalanyl-, and tyrosyl-tRNA synthetases also occurred as part of a large complex or complexes. Activity of glycyl- or leucyl-tRNA synthetase was not detected in a complex. The specific activities of arginyl- and methionyl-tRNA synthetases were three- and five-fold higher, respectively, in a complex from KA31 as compared with a complex from Balb/3T3. In contrast, the specific activity of lysyl-tRNA synthetase from the Balb/3T3 complex was 50% higher than that of the KA31 complex. tRNALys obtained from the complexes of Balb/3T3 and KA31 was fractionated into isoacceptors on columns of RPC-5. The relative amounts of lysine isoacceptors in total preparations of tRNA from normal whole cells and in tRNA obtained from the normal enzyme complex were the same. However, two isoacceptors were present in greater amounts and two were present in lesser amounts in the KA31 enzyme complex as compared with lysine isoacceptors in a total preparation of tRNA from KA31 cells.