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El virus del moteado suave del pimiento (PMMoV : estudio de la función de una glutaredoxina de "Capsicum chinense" en la interacción virus-planta

Otros títulosPepper mild mottle virus (PMMoV) : study of the role of a "Capsicum chinense" glutaredoxin in plant-virus interaction
AutorMariyappan, Saravana Kumar Ramasamy
DirectorSerra, María Teresa ; García-Luque, Isabel
Palabras claveInteracción virus-planta
Capsicum chinense
Fecha de publicación2013
Resumen[EN] Diseases caused by plant viruses are one of the major problems for agriculture production all over the world. In pepper crops (Capsicum spp), Pepper mild mottle virus (PMMoV) is one of the most destructive virus. This virus belongs to the Tobamovirus genus. In Capsicum spp, resistance against tobamovirus is governed by an allelic serie of genes: the L genes and Hk gene. Based upon the pathogen aggressiveness against L-gene mediated resistance, Tobamoviruses has been classified as pathotypes P0, P1, P1,2, P1,2,3 and P1,2,3,4. PMMoV is the most pathogenic tobamovirus towards the L resistance gene. The Italian strain of PMMoV (PMMoV-I) was used in this work. It belongs to the pathotype P1,2,3 which is able to infect systemically the L1- L3 Capsicum plants, and it is localized in the L4 plants. In our laboratory, it has been previously isolated a Glutaredoxin (Grx) from C. chinense plants whose mRNA is induced in both the compatible and the incompatible interactions after the infection with PMMoV-I and PMMoV-S, respectively. Grx are small thiol disulfide oxidoreductases that catalyze the reduction of disulfide bonds either in the target proteins or in the substrates in the presence of glutathione (GSH). Grxs are well conserved in all organisms and their function in Escherichia coli, yeast and mammal are well characterized. Since very little information was available for the function of these Grx proteins in plants and also against plant pathogens, the main aim of this work was to carry out the molecular and biochemical characterization of the C. chinense Grx (Cch Grx) protein and to analyze its function in PMMoV-I infection and/or plant response. To address these objectives the previous work from our group constructed Nicotiana benthamiana Domin transgenic plants constitutively expressing the native choroplastic Grx protein Cch Grx (Grx Exp), Cch Grx fused to GFP (Grx:GFP) and a derivative with a deletion of 63 aa at its N terminus fused to GFP (Cch Δ2M:Grx:GFP) that does not located to the chloroplasts but to the nuclei. N. benthamiana Domin plants are susceptible to all of the tobamoviruses and it is appropriate to study the role of Cch Grx in a plant-virus compatible interaction. The Italian strain of Pepper mild mottle virus (PMMoV-I) was chosen to carry out this study, because preliminary experiments from our laboratory had shown that at latest stages of the infection their constitutive expression in this host lead to a reduced viral accumulation that it was due to a reduced number of infected cells, but not to a reduced cell-to-cell movement.
By transient expression in N. benthamiana plants it was also established that the protein was located to the chloroplast, and that the 63 aa N- deleted protein was located to the nuclei. Cch Grx protein sequence analysis shows that it belongs to Group I monothiol Grx and it is positioned in the S12 subgroup with a WCSYS active site, them we referred to it as Cch GrxS12. The BLAST analysis shows that the protein belongs to single domain Grxs and further the phylogenetic tree shows that the protein is showing maximum identity with the Grxs reported from plants and much divergence with the Grx reported from yeast. CSYS active site Grxs and Grx-like proteins were found to be conserved among the plants and yeast but not reported from prokaryotes. The 3-D structural predictions for the protein show that it belongs to the Trx-fold family proteins which are characterized by having centrally located β-strands surrounded by α- helices. The structure predicted in this work for Cch GrxS12 was found to be identical to the structure reported for PtrGrxS12 from Populus. The comparison of the two Grxs structures revealed that most of the regions including the GSH binding regions were conserved between these two proteins. To characterize the oxidoreductase activity of the Cch GrxS12 protein, it was expressed and purified from E. coli and the in vitro activity of the protein was measured by HED assay, in which the oxidization of NADPH was measured. The protein was found to be positive in the HED assay and thus possesses the oxidoreductase activity. To confirm the subcellular localization of Cch GrxS12, protoplasts were obtained from the N. benthamiana transgenic lines and the GFP-fused proteins were observed by confocal microscopy, confirming that Cch GrxS12 fused to GFP was targeted to the chloroplast whereas the 63aa truncated Grx fused to GFP was located in both the cytoplasm and the nucleus, as free GFP. The Cch GrxS12 accumulation level was analyzed in the different Cch GrxS12- expressing transgenic lines by means of Western blot using Cch GrxS12 specific immunoserum available from our laboratory. The analysis showed that Cch GrxS12 accumulation was ten times higher in the N .benthamiana line expressing native Cch GrxS12 fused to GFP than in the other plant lines. The effect of the constitutive expression in N. benthamiana plants of Cch GrxS12 targeted to either the chloroplast or to the nucleus and cytoplasm on PMMoV-I viral infection was analyzed during early (7 dpi) up to late (28 dpi) stages. At 21 dpi the plants start to recover from the PMMoV-I infection. The inhibitory role of Cch GrxS12 against PMMoV-I accumulation was studied by analyzing the viral CP and RNA accumulation. Expression of Cch GrxS12 in N. benthamiana plants was found to inhibit the viral accumulation at the later stages of infection but none effect was found at the early stage of infection. The data show that Cch GrxS12 is not inhibiting the viral replication, as it was confirmed by the analysis of viral PMMoV-I infection in plant protoplasts from the different transgenic lines. It was found that viral CP accumulation was uniform at the different time points analyzed and it was also found that the percentage of infected protoplasts was the same among the GFP-expressing plant line and Cch GrxS12-expressing ones.
To study the underlying resistance mechanism mediated by Cch GrxS12 against PMMoV-I, the accumulation level of SA-regulated acidic PR protein mRNAs (PR-1, PR-2a and PR-5) and ethylene (ET)-regulated basic PR protein mRNA (PR-2b) were analyzed in the GFP expressing and Cch GrxS12 over expressing lines during early and late stage of infection. At early stages of infection, the accumulation level of SA-regulated PR mRNAs got increased in the PMMoV-I inoculated leaves when compared to the mock inoculated control leaves while the ETregulated basic PR mRNA accumulation was found to be the same between the mock-inoculated control plants and PMMoV-I inoculated ones.This shows that SA-regulated PR protein genes were induced by PMMoV-I infection whereas the ET-mediated PR protein gene was induced by mechanical injury. Further in the inoculated and in the systemic leaves, no difference in PR mRNAs accumulation exists between Cch GrxS12 over expressing lines and GFP expressing line. At late stage of PMMoV-I infection (21 and 28 dpi), the accumulation level of SA-regulated PR mRNAs was found to be higher than the ethylene-regulated PR mRNA. In addition, in the mock-inoculated control plants, over-expression of Cch GrxS12 leads to the accumulation of a high level of SA-regulated PR mRNAs and it was found to be higher than that of the ETregulated PR one. Further, in the PMMoV-I infected plants, the over-expression of Cch GrxS12 enhanced the accumulation level of SA-regulated PR mRNAs when compared to the GFP expressing line. Cch GrxS12 was found to have little effect in ET-regulated PR mRNA. At 28 dpi, analysis of the recovered plant leaves shows a high level of PR mRNA accumulation in all of the infected plants, being higher in the Grx expressing plant lines. To analyze whether Cch GrxS12 was able to induce one or more defence pathway, the mRNA expression level of 9-LOX, which is independent of JA and SA pathways, was analyzed. At 7 dpi, in the PMMoV-I inoculated plants an increase in 9-LOX mRNA accumulation was found in the Cch GrxS12 expressing lines while at 28 dpi the increase was found in the recovered leaves which shows that Cch GrxS12 over-expression could be able to activate more than one plant defence response pathway during the plant development and also during PMMoVI infection. Analysis of the antioxidant enzyme machinery of the cell system such as GR and GST were also found to get vary along the PMMoV-I infection. GR mRNA accumulation got decreased in the PMMoV-I infected plants and the expression of Cch GrxS12 was not found to alter the expression of GR mRNA. The accumulation level of GST mRNA was found to get increased during the PMMoV-I infection at 7 and 21 dpi but at 28 dpi, the accumulation level got reduced in the Cch GrxS12 expressing lines what suggests that Cch GrxS12 could be able to reduce the oxidative stress conditions caused by the PMMoV-I infection at that time. To analyze the role of Cch GrxS12 in maintaining the redox status of the cell, and also to have better insight in the Grx-mediated PR mRNA accumulation, the oxidized and reduced forms of the pyridine nucleotides (PN) [NAD+, NADH, NADP+ and NADPH] were analyzed at the late stage of plant growth (21 and 28 dpi) in the control and Cch GrxS12 over-expressing lines. . Overall the level of NAD+, NADH, NADP+ contents got increased at 28 dpi when compared with 21 dpi whereas the level of NADPH was found to be lowered. Analysis of the PNs in the mock-inoculated control plants shows that expression of Cch GrxS12 does not seemingly alter the redox status of these metabolites as their level were found to be the same between the control and Cch GrxS12-over expressing lines.
On the other hand, infection with PMMoV-I altered the PNs levels, where the accumulation level of the reduced form of the PNs such as NADH and NADPH got increased when compared with the mockinoculated control plants. The accumulation level of NADH and NADPH was particularly higher in the Nb:Grx-GFP line where the Grx accumulation was also found to be higher. Among the reduced PNs, the level of NADH content was found to be 4-8 times higher in the PMMoV-I infected plants and thus revealed that NADH content is a good marker for PMMoV-I infection. Comparative analysis of the PNs between the mock-inoculated controls, PMMoV-I infected and recovered plant samples shows that alteration in the PNs level is not influencing the PR mRNAs accumulation and thus suggesting that Cch GrxS12-mediated PR mRNAs accumulation is independent of PN alteration.
Descripción155 p.- 31 fig.- 3 tab.
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