M. T. Serra

Nucleotide sequence of the genomic RNA of pepper mild mottle virus, a resistance-breaking tobamovirus in pepper

The entire genomic RNA of a Spanish isolate of pepper mild mottle virus (PMMV-S), a resistance-breaking virus in pepper, was cloned and sequenced and shown to be similar to other tobamoviruses in its genomic organization. It consisted of 6357 nucleotides (nt) and contained four open reading frames (ORFs) which encode a 126K protein and a readthrough 183K protein (nt 70 to 4908), a 28K protein (nt 4909 to 5682) and a 17.5K coat protein (nt 5685 to 6158). This is the first tobamovirus in which none of the ORFs overlap. Both its nucleic acid and predicted protein sequences were compared with the previously determined sequences of other tobamoviruses. The variations and similarities found and their relationship with the pathogenicity of this virus are discussed.

Proteomic analysis of pathogenesis-related proteins (PRs) induced by compatible and incompatible interactions of pepper mild mottle virus (PMMoV) in Capsicum chinense L3 plants.

Resistance conferred by the L(3) gene is active against most of the tobamoviruses, including the Spanish strain (PMMoV-S), a P(1,2) pathotype, but not against certain strains of pepper mild mottle virus (PMMoV), termed P(1,2,3) pathotype, such as the Italian strain (PMMoV-I). Both viruses are nearly identical at their nucleotide sequence level (98%) and were used to challenge Capsicum chinense PI159236 plants harbouring the L(3) gene in order to carry out a comparative proteomic analysis of PR proteins induced in this host in response to infection by either PMMoV-S or PMMoV-I. PMMoV-S induces a hypersensitive reaction (HR) in C. chinense PI159236 plant leaves with the formation of necrotic local lesions and restriction of the virus at the primary infection sites. In this paper, C. chinense PR protein isoforms belonging to the PR-1, beta-1,3-glucanases (PR-2), chitinases (PR-3), osmotin-like protein (PR-5), peroxidases (PR-9), germin-like protein (PR-16), and PRp27 (PR-17) have been identified. Three of these PR protein isoforms were specifically induced during PMMoV-S-activation of C. chinense L(3) gene-mediated resistance: an acidic beta-1,3-glucanase isoform (PR-2) (M(r) 44.6; pI 5.1), an osmotin-like protein (PR-5) (M(r) 26.8; pI 7.5), and a basic PR-1 protein isoform (M(r) 18; pI 9.4-10.0). In addition, evidence is presented for a differential accumulation of C. chinense PR proteins and mRNAs in the compatible (PMMoV-I)-C. chinense and incompatible (PMMoV-S)-C. chinense interactions for proteins belonging to all PR proteins detected. Except for an acidic chitinase (PR-3) (M(r) 30.2; pI 5.0), an earlier and higher accumulation of PR proteins and mRNAs was detected in plants associated with HR induction. Furthermore, the accumulation rates of PR proteins and mRNA did not correlate with maximal accumulation levels of viral RNA, thus indicating that PR protein expression may reflect the physiological status of the plant.

The Coat Protein Is Required for the Elicitation of the Capsicum L2 Gene-Mediated Resistance Against the Tobamoviruses

In Capsicum, the resistance against tobamoviruses conferred by the L2 gene is effective against all but one of the known tobamoviruses. Pepper mild mottle virus (PMMoV) is the only virus which escapes its action. To identify the viral factors affecting induction of the hypersensitive reaction (HR) mediated by the Capsicum spp. L2 resistance gene, we have constructed chimeric viral genomes between paprika mild mottle virus (PaMMV) (a virus able to induce the HR) and PMMoV. A hybrid virus with the PaMMV coat protein gene substituted in the PMMoV-S sequences was able to elicit the HR in Capsicum frutescens (L2L2) plants. These data indicate that the sequences that affect induction of the HR mediated by the L2 resistance gene reside in the coat protein gene. Furthermore, a mutant that codes for a truncated coat protein was able to systemically spread in these plants. Thus, the elicitation of the host response requires the coat protein and not the RNA.

Characterization of a pathogenesis-related protein 4 (PR-4) induced in Capsicum chinense L3 plants with dual RNase and DNase activities

Resistance conferred by the L3 gene is active against most of the tobamoviruses, including the Spanish strain (PMMoV-S), a P1,2 pathotype, but not against certain strains of pepper mild mottle virus (PMMoV), termed as P1,2,3 pathotype, such as the Italian strain (PMMoV-I). PMMoV-S induces a hypersensitive reaction (HR) in C. chinense PI159236 plant leaves with the formation of necrotic local lesions and restriction of the virus at the primary infection sites. In this paper, a C. chinense PR-4 protein induced during both the compatible and the incompatible interactions has been identified. It was strongly associated with HR induction and to a lesser extent with the compatible interaction, but only in the later stages of infection. Moreover, it was found to accumulate during the necrogenic reaction induced by Potato virus X. The C. chinense PR-4 protein belongs to the PR-4 protein subgroup II, based on the absence of a hevein domain. Furthermore, it is shown that the purified protein does not have chitinase activity, as previously proposed for PR-4 proteins. Instead, it has both RNase and DNase activity, although its contribution to the bulk activity of nucleases in infected plants is very low.

Pepper Mild Mottle Virus Coat Protein Alone Can Elicit the Capsicum spp. L3 Gene-Mediated Resistance

The pepper mild mottle virus (PMMoV-S) (an L3 hypersensitive response [HR]-inducer strain) coat protein was expressed in Capsicum chinense (L3L3) plants with the heterologous potato virus X (PVX)-based expression system. The chimeric virus was localized in the inoculated leaves and induced the HR, thus indicating that the tobamoviral sequences that affect induction of the HR conferred by the L3 resistance gene reside in the coat protein gene. Furthermore, transient expression of the PMMoV-S coat protein in C. chinense leaves by biolistic co-bombardment with a plasmid expressing the β-glucuronidase (GUS) gene leads to the induction of cell death and expression of host defense genes. Thus, the coat protein of PMMoV-S is the elicitor of the Capsicum spp. L3 resistance gene-mediated HR.

Altered local and systemic spread of movement deficient virus in transgenic tobacco plants expressing the cucumber mosaic virus 3a protein.

Summary. The 3a protein encoded by RNA 3 of cucumber mosaic virus has been identified as the viral cell-to-cell movement protein. The constitutive expression in transgenic tobacco plants of 3a protein from a subgroup I strain was able to complement in trans the short distance movement of a 3a defective CMV mutant belonging to a different taxonomic subgroup. This ability was dependent upon the accumulation levels of the 3a protein in transgenic tobacco plants. However, an initial delay in viral accumulation and spread of the defective virus as compared to the wild type virus was determined in complementation tests. Furthermore, a reduction in disease symptoms as well as a different pattern of systemic viral distribution from those of the wild type virus was detected. These results show that the early events in viral infection affect the long distance spread of the virus. Finally, the wild type virus moved faster in the 3a protein-expressing plants than in control plants, thus indicating that the constitutive expression of the 3a protein favours long-distance viral spread.

Accumulation kinetics of CMV RNA 3-encoded proteins and subcellular localization of the 3a protein in infected and transgenic tobacco plants

SummaryThe complete nucleotide sequence of RNA 3 of a Spanish isolate of cucumber mosaic virus (CMV-24) has been determined. The encoded putative cell-to-cell movement protein (3a protein) and the coat protein are 279 and 218 amino acids long, respectively. The 3a protein was expressed inEscherichia coli using the vector pT7-7 and was used to raise an immunoserum. We have followed the time course of accumulation of the 3a protein, in parallel to that of the coat protein, and its subcellular localization as a function of time after CMV-24 infection on tobacco plants. The maximum accumulation level of the 3a protein was reached at early stages of infection, being detected in the cytosolic and the cell wall fractions. At later stages of infection, a decline in accumulation levels of the 3a protein was observed, and the protein was essentially associated with the cell wall fractions. These data were corroborated by immunocyto-chemistry performed in both infected and 3a-expressing transgenic tobacco plants.

Development and use of detection methods specific for Cucumber vein yellowing virus (CVYV)

Two methods for the detection of Cucumber vein yellowing virus (CVYV) on infected plants were developed, based on the information provided by cDNA clones covering the 3′-end of the genome of a Spanish isolate (CVYV-AILM). The sequenced portion of the CVYV-AILM genome showed a 96.6% aminoacid identity with that of a reported sequence of another CVYV isolate from Israel (Lecoq et al., 2000). The first detection method used a RNA specific probe for hybridization with nucleic acids extracted from infected plants. The probe was complementary to a portion of the CVYV genome including the C-terminal part of the NIb and most of the coat protein (CP) coding regions. The second detection method employed polyclonal antisera raised against recombinant viral CP expressed in bacteria. The specific antibodies were used to detect the presence of virus particles in plant extracts. Both procedures resulted in a highly specific detection of CVYV in plants infected with different isolates of the virus. No interference was observed with other cucurbit-infecting viruses. Sensitivities achieved were sufficient for routine diagnosis of the presence of the virus in plants.

Biological and molecular characterization of P101 isolate, a tobamoviral pepper strain from Bulgaria

Summary. A tobamovirus isolated from pepper crops in Bulgaria has been characterized, and is referred to below as P101. It was closely related to Paprika mild mottle virus (PaMMV) (Dutch isolate), based upon the serological relationship of its coat protein, and the nucleotide sequence analysis of the gene encoding the coat protein and the 3′ non-coding region of the viral RNA. The coat proteins of the two isolates differ by two amino acids, and these substitutions may be responsible for the different reactivity of the isolates towards a polyclonal antiserum raised against the virion of the Dutch isolate.The biological behaviour of both isolates was similar in the hosts tested, except in pepper plants where P101 induced delayed and milder symptoms compared with PaMMV, although their accumulation levels were similar. In addition, we investigated the infection pattern of the two isolates in tomato plants. Both isolates accumulated in protoplasts as well as in inoculated leaves, although systemic invasion was limited. This limited spread was not due to activation of defense mechanism(s) in the plant, since the upper uninoculated leaves from P101-infected tomato plants were fully susceptible to challenge inoculation with the virus. Instead, it appears due to a restriction of long-distance movement, that could be overcome in tomato plants co-infected with Tobacco mosaic virus (TMV), but not with either Cucumber mosaic virus or Pepino mosaic virus. The ability of P101 to move systemically in the presence of TMV was not linked to enhanced accumulation of P101 at the cellular level. Thus, a tobamovirus but not the viruses tested from other genera could complement, in trans, the function(s) required for PaMMV to invade the upper uninoculated leaves. Paprika mild mottle virus strain B is proposed as the name for this new isolate.

Pepper resistance-breaking tobamoviruses: Can they co-exist in single pepper plants?

We previously reported a procedure for identifying pathotypes of the tobamoviruses infecting the L-resistant genotypes of pepper based on the polymerase chain reaction (PCR) (Tenllado et al., 1994). We have now used this method to assess pathotype incidence in virus isolates from field pepper samples representing three successive epidemic episodes in southeastern Spain, and to analyse the interaction of pathotypes during experimental infections in pepper plants. The majority of virus isolates corresponded to the P1,2 pathotype, and only three out of twenty behaved as P1,2,3 pathotypes. Interestingly, restriction enzyme analysis of the PCR-amplified products distinguished two restriction subgroups on each P1,2 and P1,2,3 pathotype, referred to as restrictopatterns I1 and I2 or II1 and II2, respectively. Experimental coinoculations of pepper plants with mixtures of two different pathotypes showed coexistence between them when inoculations proceeded simultaneously. However, reciprocal cross-protection was observed between P1,2 and P1,2,3 pathotypes when they were successively inoculated, while no cross-protection was observed between P1 and either P1,2 or P1,2,3 pathotypes. The potential of the PCR-based method for detecting heterogeneity within P1,2 and P1,2,3 pathotypes, and the possibility of genetically engineered resistance to those viruses by genetic transformation with viral coat protein genes are briefly discussed.