Yoshio Ehara

Cauliflower mosaic virus gene VI causes growth suppression, development of necrotic spots and expression of defence-related genes in transgenic tobacco plants

SummaryIn order to study possible functions of the inclusion body matrix protein (IBMP) encoded by gene VI of cauliflower mosaic virus (CaMV), the XbaI fragment containing the gene VI of a Japanese strain of CaMV (CaMV S-Japan) was transferred to tobacco plants by Ti mediated transformation. Eight out of 18 kanamycin resistant plants (40%) expressed detectable levels of IBMP. Those transgenic plants expressing IBMP produced leaves with light green color, and their growth was suppressed as compared with control plants. Symptom-like necrotic spots also appeared on the leaves and stems of the mature transgenic plants. Furthermore, in these transgenic plants, pathogenesis-related proteins 1a, 1b and 1c were highly expressed and the activity of 1,3-β-glucanase was increased up to eightfold. From these results, we concluded that expression of the IBMP is associated with symptom development.

Comparison of the in vitro Translation Products of Wild-type and a Deletion Mutant of Soil-borne Wheat Mosaic Virus

Summary A wild-type isolate (WT) of soil-borne wheat mosaic virus (SBWMV, isolate JT) (RNA I 6·9 kb and RNA II 3·6 kb) was successively transferred to wheat plants by mechanical inoculation and a deletion mutant (DM) (RNA I 6·9 kb and RNA II 2·1 kb) was produced. In wheat germ extracts, RNA I of both WT and DM directed synthesis of polypeptides having mol. wt. of 220000 (220K) and 150K, and RNA II of both WT and DM directed synthesis of 25K and 19K polypeptides. Incubation in wheat germ extracts of WT or DM virions purified with an alkaline buffer also gave 220K, 25K and 19K polypeptides as major products and a 150K polypeptide as a minor product. In rabbit reticulocyte lysates, RNA I of both WT and DM directed synthesis of only the 220K polypeptide, whereas WT RNA II produced 100K, 46K, 25K and 19K polypeptides and DM RNA II, 31K, 25K and 19K polypeptides. SBWMV DM antiserum precipitated polypeptides of 100K, 31K, 25K and 19K but not 220K and 46K. The 19K polypeptide was identified as the capsid protein from its electrophoretic mobility and its reaction with antiserum to virus particles. Thus, the differences between the in vitro translation products of WT and DM SBWMV were confined only to those coded by RNA II.

A protein in the oxygen-evolving complex in the chloroplast is associated with symptom expression on tobacco leaves infected with cucumber mosaic virus strain Y

To elucidate the molecular basis of symptom expression in virus-infected plants, the changes in proteins between tobacco, Nicotiana tabacum cv. Ky57, leaves inoculated with cucumber mosaic virus strain Y [CMV(Y)] and strain O [CMV(O)], were compared by 2-dimensional (2-D) gel electrophoresis. The appearance of chlorotic spots in CMV(Y)-inoculated tobacco leaves accompanied an increase of 3 polypeptides and a decrease in 6 polypeptides, as compared with those in the CMV(O)-inoculated tobacco which showed no clear symptoms. The decrease in the amounts of two polypeptides of 22 and 23 kDa was particularly significant: these two polypeptides were compared with a 24 kDa polypeptide, which co-migrated with them in 2-D gel electrophoresis but did not clearly decrease at an early stage of infection, as well as major other proteins of CMV(Y)-inoculated tobacco leaves. However, the 22, 23 and 24 kDa polypeptides showed the same peptide mapping pattern. Furthermore, the 12 amino acid residues at N-termini of the three polypeptides match those of the extrinsic 23 kDa polypeptide of an oxygen-evolving complex from spinach. A comparative analysis of the 22, 23 and 24 kDa polypeptides in N. tabacum and its ancestral parents, N. sylvestris and N. tomentosiformis, revealed that the 22 kDa polypeptide derives from N. sylvestris and the 23 kDa polypeptide from N. tomentosiformis; the 24 kDa polypeptide derives from both ancestral Nicotiana species. The results indicate that the polypeptides whose amounts differentially decrease with the progress of symptom expression in N. tabacum inoculated with CMV(Y) are one component of the oxygen-evolving complex in photosystem II.

One Amino Acid Change in Cucumber Mosaic Virus RNA Polymerase Determines Virulent/Avirulent Phenotypes on Cowpea

ABSTRACT The elicitation of the hypersensitive response (HR) is known to depend on the interaction between a resistance gene of a host plant and a corresponding avirulence gene of a pathogen. The cv. Kurodane-Sanjaku of cowpea (Vigna unguiculata) has the Cry locus that confers resistance against cucumber mosaic virus strain Y (CMV-Y). The resistance is overcome by infection with a legume strain of CMV (CMV-L). RNA 2, which codes for the 2a protein, a subunit of the viral replicase components, has been known to control virulent/avirulent phenotypes. We generated chimeric constructs of full-length cDNA clones of RNA 2 of both strains and inoculated infectious transcripts to delimit the domain controlling symptoms. A 243-base pair fragment containing a coding region for the GDD RNA-dependent RNA polymerase core sequence was shown to be responsible for the phenotypic differences. From sequence alignment analysis, two amino acids (Phe631 and Ala641) of the HR-type 2a protein encoded in this fragment were specifically exchanged to Tyr and Ser, respectively, in the 2a proteins of resistance-breaking strains. Point mutations introduced into RNA 2 backgrounds of both strains that were designed to change the amino acid at position 631 resulted in a change of symptoms, indicating that a single nucleotide change determines the reactions elicited by both strains. Analysis for one additional mutant RNA 2 showed that symptom determination may be correlated with the nature of the lateral chain of amino acid 631.

Nucleotide sequence analyses of peanut stunt cucumovirus RNAs 1 and 2

The nucleotide sequences of the RNAs 1 and 2 of the peanut stunt virus strain J (PSV-J) were determined and compared with those of the cucumber mosaic virus strain Y (CMV-Y, subgroup I), strain Q (CMV-Q, subgroup II) and the tomato aspermy virus strain V (TAV-V) at both the nucleotide and protein levels. RNA 1 of PSV-J consists of 3355 nucleotides (nt) and has one large open reading frame (ORF) which can encode the putative 1a protein of Mr 112025. PSV-J RNA 1 and the 1a protein are 65 to 73% identical to those of CMV-Y and -Q, and 65 to 69% to those of TAV-V, RNA 2 of PSV-J contains 2946 nt and also has one large ORF which can encode the putative 2a protein of Mr 93575. For RNA 2 and the 2a protein, identities between PSV-J and two strains of CMV are calculated to be 53 to 61%. When compared with TAV-V, the same degree of similarity as seen with CMVs is observed. The 1a protein has the consensus sequences found in some helicases and methyltransferases and the 2a protein includes a sequence which exists in several RNA-dependent RNA polymerases.

Nucleotide sequence of RNA 3 of peanut stunt cucumovirus

The nucleotide sequence of the RNA 3 of the peanut stunt virus strain J (PSV-J) was determined and compared with those of the cucumber mosaic virus strain Y (CMV-Y, subgroup I) and the tomato aspermy virus strain C (TAV-C) at both the nucleotide and protein levels. The RNA 3 of PSV-J consists of 2186 nucleotides and has two large open reading frames (ORFs). By analogy to other tripartite plant viruses, it is presumed that the first ORF (867 nucleotides) codes for the 3a protein and the second ORF (654 nucleotides) is the cistron of the coat protein which is expressed from subgenomic RNA 4 of 1010 nt residues. At the nucleotide level, PSV-J RNA 3 shows 60.6% homology to RNA 3 of CMV-Y and 66.3% to that of TAV-C, much lower than the value between the two CMV subgroups. For the 3a proteins there is 65.4% homology between PSV-J and CMV-Y and 70.2% homology between PSV-J and TAV-C. Although it has been known that PSV shows a distant serological relationship to CMV, the coat protein of PSV-J shows only 50% homology with that of CMV-Y.

Distribution of Cylindrical Inclusion, Amorphous Inclusion and Capsid Proteins of Watermelon Mosaic Virus 2 in Systemically Infected Pumpkin Leaves

Summary Monoclonal antibodies (MAbs) were prepared against the cylindrical inclusion protein (CIP), amorphous inclusion protein (AIP) and capsid protein (CP) of watermelon mosaic virus 2 (WMV2). Using the MAbs, CIP, AIP and CP were detected, roughly quantitatively, in WMV2-infected pumpkin leaves showing various symptoms by using electroblot-ELISA. From symptomless leaves and most dark green areas in the mosaic pattern no protein or small amounts of the three proteins were detected, but from most yellow areas in the mosaic almost equal amounts of each protein were detected in abundance. Leaves showing mild vein-yellowing and vein-clearing (respectively, the first and second leaves of the plants tested) contained AIP and CP in large amounts, but little CIP. On the other hand, expanding leaves contained CIP and AIP in large quantities, but CP in traces only. Therefore the distributions of CIP, AIP and CP in pumpkin plants were very uneven, but correlated with symptoms. In addition, the ratio of the concentrations of CIP, AIP and CP varied from tissue to tissue.

Activation of defense-related gene expression and systemic acquired resistance in cucumber mosaic virus-infected tobacco plants expressing the mammalian 2'5'oligoadenylate system.

Summary. Tobacco plants expressing the mammalian 2′5′oligoadenylate system (2-5A system) exhibit resistance to cucumber mosaic virus (CMV). Here, to characterize the molecular aspect of the resistance to CMV in 2-5A system-expressing tobaccos, the activation of defense-related genes and systemic acquired resistance (SAR) as the markers for the hypersensitive resistance (HR), were elucidated. Northern hybridization analysis indicated that the expression of four pathogenesis-related (PR) protein genes and five HR-related genes were induced in CMV-infected tobaccos expressing 2-5A system. Furthermore, the induction of SAR against Pseudomonas syringae pv. tabaci as second challenge, was observed on CMV-inoculated tobaccos expressing 2-5A system. These results suggested that the resistance to CMV in tobacco expressing 2-5A system is associated with the establishment of an HR-like response.

Isolation and serological comparison of virus-coded proteins of three potyviruses infecting cucurbitaceous plants

Cylindrical inclusion proteins (CIPs), amorphous inclusion proteins (AIPs), and virus particles were partially purified from Cucurbita maxima leaves infected with zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus 2 (WMV2), and the watermelon mosaic virus 1 strain of papaya ringspot virus (PRSV-W). Antisera to these individual antigens, except AIPs of ZYMV and WMV2, were prepared and used to determine with electroblot-ELISA the serological differentiation indices (SDIs) between homologous and heterologous antigens. The average SDIs between ZYMV and WMV2 CIPs, between ZYMV and PRSV-W CIPs, and between WMV2 and PRSV-W CIPs were 1.5, 3.5, and 2.5, respectively. The SDIs between ZYMV and PRSV-W AIPs and between WMV2 and PRSV-W AIPs were 5 and 3, respectively. The average SDIs between ZYMV and WMV2 capsid proteins (CPs), between ZYMV and PRSV-W CPs, and between WMV2 and PRSV-W CPs were 3, 7, and 8, respectively. These data suggest that the serological relationship between ZYMV and WMV2 is much closer than that between PRSV-W and ZYMV or between PRSV-W and WMV2 and that antigenic determinants of CIP and AIP were conserved more than those of CP among the three potyviruses.

Biological control of soft rot of Chinese cabbage using single and mixed treatments of bacteriocin-producing avirulent mutants of Erwinia carotovora subsp. carotovora.

Two pathogenic strains of E. carotovora subsp. carotovora 2T-2 and TT-4 with high bacteriocin activity but low sensitivity to the bacteriocins of other strains were treated with ethyl methane sulphonate (EMS). Two avirulent mutants A-f-39 and B-e-19 developed from 2T-2 and TT-4, respectively, by this treatment had the same bacteriocin activity as their respective parents and inhibited the in vitro growth of pathogenic strains of this species. The disease control of these two mutant strains were compared in the field in 1995 and 1997 to the control by CGE234M403 (M403) (a commercialized biocontrol agent), a mixture of A-f-39 and M403, and an agrochemical (basic dithianon-copper chloride). The protection obtained with A-f-39 was comparable to M403 and was better than that with the chemical. The mixture of A-f-39 and M403 consistently gave the best results in all the field trials.