The effect of viral infection on photosynthesis was investigated infected plants. The CP accumulation level was dependent upon both the post-infection time and the virus analyzed, but in Nicotiana benthamiana Gray plants infected with different strains of pepper and paprika mild mottle viruses (PMMoV independent of the CP itself since hybrid viruses did not behave as their parental viruses with the same CP, with and PaMMoV) and chimeric viral genomes derived from them. In both symptomatic and asymptomatic leaves of virus-respect to PSII inhibition, CP accumulation rates and OEC infected plants, photosynthetic electron transport in photosys-protein levels. Modulated chlorophyll (Chl) fluorescence and tem II (PSII) was reduced. In all cases analyzed, viraloxygen evolution measurements carried out in both types of infection affected the polypeptide pattern of the oxygen-evolv-leaves showed that the quantum yield of PSII electron transport was diminished in infected plants with respect to those of ing complex (OEC) in thylakoid membranes. The levels of control plants. The decrease in electron transport efficiency both the 24 and 16 kDa proteins were reduced to a differing extent when compared with the levels in healthy control. This was mainly caused by a reduction in the fraction of open loss of the OEC extrinsic proteins affected the oxygen evolu-reaction centers. The infected plants also showed a reduction tion rates of thylakoid membranes and leaves from infected in the efficiency of excitation capture in PSII by photoprotecplants. Additionally, viral coat protein (CP) was found associ-tive thermal dissipation of excess excitation energy. ated with the chloroplasts and the thylakoid membranes of the phenomenon under biotic stress, as is also observed under abiotic stress conditions. However, the mechanism of action of the viral infection upon PSII remains unclear (see Balachandran et al. 1997).Chlorophyll (Chl) fluorescence measurements show that the PSII photochemical efficiency was decreased by the viral infection (Hodgson et al. 1989, van Kooten et al. 1990, Balachandran and Osmond 1994, Balachandran et al. 1994a). Studies of imaging PSII fluorescence quenching (Balachandran et al. 1994b) also demonstrated that in the youngest, most susceptible leaves of tobacco plants systemically infected with tobbaco mosaic virus (TMV), distur-

We have analyzed promoter regulatory elements from a photoregulated CAB gene (Cab‐E) isolated from Nicotiana plumbaginifolia. These studies have been performed by introducing chimeric gene constructs into tobacco cells via Agrobacterium tumefaciens‐mediated transformation. Expression studies on the regenerated transgenic plants have allowed us to characterize three positive and one negative cis‐acting elements that influence photoregulated expression of the Cab‐E gene. Within the upstream sequences we have identified two positive regulatory elements (PRE1 and PRE2) which confer maximum levels of photoregulated expression. These sequences contain multiple repeated elements related to the sequence‐ACCGGCCCACTT‐. We have also identified within the upstream region a negative regulatory element (NRE) extremely rich in AT sequences, which reduces the level of gene expression in the light. We have defined a light regulatory element (LRE) within the promoter region extending from −396 to −186 bp which confers photoregulated expression when fused to a constitutive nopaline synthase (‘nos’) promoter. Within this region there is a 132‐bp element, extending from −368 to −234 bp, which on deletion from the Cab‐E promoter reduces gene expression from high levels to undetectable levels. Finally, we have demonstrated for a full length Cab‐E promoter conferring high levels of photoregulated expression, that sequences proximal to the Cab‐E TATA box are not replaceable by corresponding sequences from a ‘nos’ promoter. This contrasts with the apparent equivalence of these Cab‐E and ‘nos’ TATA box‐proximal sequences in truncated promoters conferring low levels of photoregulated expression.

We have studied by kinetic Chl-fluorescence imaging (Chl-FI) Nicotiana benthamiana plants infected with the Italian strain of the pepper mild mottle tobamovirus (PMMoV-I). We have mapped leaf photosynthesis at different points of the fluorescence induction curve as well as at different post-infection times. Images of different fluorescence parameters were obtained to investigate which one could discriminate control from infected leaves in the absence of symptoms. The non-photochemical quenching (NPQ) of excess energy in photosystem II (PSII) seems to be the most adequate chlorophyll fluorescence parameter to assess the effect of tobamoviral infection on the chloroplast. Non-symptomatic mature leaves from inoculated plants displayed a very characteristic time-varying NPQ pattern. In addition, a correlation between NPQ amplification and virus localization by tissue-print was found, suggesting that an increase in the local NPQ values is associated with the areas invaded by the pathogen. Changes in chloroplast ultrastructure in non-symptomatic leaf areas showing different NPQ levels were also investigated. A gradient of ultrastructural modifications was observed among the different areas.

We have previously shown that tobamovirus infection induces an inhibition of photosystem II electron transport, disturbing the oxygen-evolving complex (OEC). In the infected plants, the OEC polypeptide pattern was modified when compared to healthy plants, the levels of the PsbP and PsbQ extrinsic proteins being lowered to different extents. In this work we have further investigated by two-dimensional polyacrylamide gel electrophoresis (2-DE) the changes on the OEC protein pattern of thylakoid membranes isolated from Nicotiana benthamiana Domin plants infected with the Spanish strain of pepper mild mottle virus. When the thylakoid membranes from healthy plants were analyzed for the presence of PsbO and PsbP proteins by 2-DE (pI range 4-7) and further immunoassayed by using specific-antisera against these two proteins, it was observed that four polypeptides cross-reacted with each antiserum. These data, along with the N-terminal amino acid sequence determined for the eight polypeptides, indicate that the N. benthamiana PsbO and PsbP proteins correspond to protein families. In the silver-stained 2-DE gels of thylakoid membranes isolated at different days postinoculation from virus-infected plants, it was observed that the content of PsbP polypeptides decreased dramatically with respect to those of PsbO, during the progress of the infection. Interestingly, there was a differential decrease of the different PsbP proteins, indicative of a distinct regulation of their expression.

The L3 gene is responsible for the hypersensitive response in Capsicum plants against infection by tobamoviruses. The resistance conferred by this gene is one of the most effective so far described against tobamoviruses. Certain isolates of pepper mild mottle virus (PMMV) are the only tobamoviruses able to overcome the L3 resistance. Chimeric viral genomes between PMMV-S (to which L3 plants are hypersensitive) and PMMV-I (an L3 resistance-breaking isolate) led us to conclude that sequence variation within the coat protein gene of both isolates determines their different virulence in L3L3 plants. Furthermore, the results indicate that a single amino acid substitution, Asn to Met, at position 138 of the PMMV-I coat protein is sufficient to induce the hypersensitive response and localization of viral infection in C. chinense plants. Finally, the use of a mutant coding for a truncated coat protein (maintaining the Met138 coding sequence at the RNA level) demonstrates that a functional coat protein is required for elicitation of the L3 gene-mediated resistance.

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.

Overcoming host resistance in gene-for-gene host-virus interactions is an important instance of host range expansion, which can be hindered by across-host fitness trade-offs. Trade-offs are generated by negative effects of host range mutations on the virus fitness in the original host, i.e., by antagonistic pleiotropy. It has been reported that different mutations in Pepper mild mottle virus (PMMoV) coat protein result in overcoming L-gene resistance in pepper. To analyze if resistance-breaking mutations in PMMoV result in antagonistic pleiotropy, all reported mutations determining the overcoming of L 3 and L 4 alleles were introduced in biologically active cDNA clones. Then, the parental and mutant virus genotypes were assayed in susceptible pepper genotypes with an L ؉ , L 1 , or L 2 allele, in single and in mixed infections. Resistance-breaking mutations had pleiotropic effects on the virus fitness that, according to the specific mutation, the host genotype, and the type of infection, single or mixed with other virus genotypes, were antagonistic or positive. Thus, resistance-breaking mutations can generate fitness trade-offs both across hosts and across types of infection, and the frequency of host range mutants will depend on the genetic structure of the host population and on the frequency of mixed infections by different virus genotypes. Also, resistance-breaking mutations variously affected virulence, which may further influence the evolution of host range expansion. Changes in virus host range affect virus ecology and epidemiology, condition virus emergence, and can compromise the success of strategies for the control of viral diseases (1-4). The acquisition of new hosts, that is, host range expansion, would provide a virus with more opportunities for transmission and survival. However, differential host-associated selection may result in across-host fitness trade-offs so that increasing the virus fitness in a new host will decrease its fitness in the original one, which will hinder host range expansion (4-6). The simplest mechanism generating across-hosts fitness trade-offs is antagonistic pleiotropy, in which mutations that increase fitness in one host are deleterious in another one (7). Evidence indicates that antagonistic pleiotropy is common in RNA viruses as a result of their small genomes, which are compacted with genetic information and encode few, multifunctional proteins (5,8).Host range evolution is particularly relevant for the sustainable use of genetic resistance to control viral diseases of crops. Disease control based on resistant cultivars is target specific and highly efficient but is not durable due to the appearance of new virus genotypes able to infect otherwise resistant host genotypes. The increase in frequency of resistance-breaking virus genotypes eventually renders resistance inefficient (9-11). Plant-virus interactions can often be explained by the gene-for-gene (GFG) model, under which direct or indirect recognition of viral proteins by those encoded by plant resistan...

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.