Carvalho, Marisa

Choupina, Altino

Cravador, Alfredo

Jorge, Lurdes

Meirinho, Sofia G.

Sousa, Maria João

Biblioteca Digital do IPB

IDENTIFICATION AND CHARACTERIZATION OF MOLECULAR FACTORS ASSOCIATED WITH THE Phytophthora cinnamomiINFECTION MECHANISMS Altino Choupina (1,2), Sofia Meirinho(1), Marisa Carvalho(1), Lurdes Jorge (1), M. João Sousa (1) and Alfredo Cravador(3)(1) Instituto Politécnico de Bragança, Escola Superior Agrária, Campus de Santa Apolónia - Apartado 1172, 5301-854 BRAGANÇA, PORTUGAL (2) CIMO Campus de Santa. Apolónia - Apartado 1172, 5301-854 BRAGANÇA, PORTUGAL (3) Universidade do Algarve (UAlg), Campus da Penha, Estrada da Penha. 8005-139 FARO,  PORTUGALMATERIALS AND METHODSAll genes referred in this study were obtained from P. cinnamomi strain Pr120 genomic DNA. Elucidation of complete gene nucleotide sequences of TGase, GIP and NPP1were achieved by High-Efficiency Thermal Asymetric Interlaced PCR (HE-TAIL PCR), a method described by Michiels et al. (2003). DNA sequencing was performed using anABI 373 automated sequencer. The open reading frames (ORF) of P. cinnamomi genes were identified by BioEdit program and submitted to EMBL databases (GIP-accessionnumber CAJ90742.1; P. cinnamomi transglu gene for transglutaminase elicitor precursor, accession AM403129; P. cinnamomi npp1 gene for necrosis-inducing protein -accession AM403130). Nucleotide and amino acid sequences were analyzed using FASTA programs from EMBL databases. ClustalW2 (Larkin et al., 2007) was used to alignthe Phytophthora genus sequences.INTRODUCTIONPhytophthora species are the causal agents of many serious plant diseases. They secrete large amounts of elicitins, a group of unique highly conserved proteins that are able toinduce hypersensitive response (HR) and enhance plant defense responses in a systemic acquired resistance (SAR) manner against infection by different pathogens.Phytophthora cinnamomi, one of the most destructive species of Phytophthora genus is the causal agent of Castanea sativa ink disease, and has been associated with thedecline of several forest, ornamental and fruit trees and shrubs, causing enormous economic losses worldwide.We briefly describe some of the proteins involved in P. cinnamomi infection mechanisms: a transglutaminase (TGase, induction of defense responses and disease-like symptoms),a glucanase inhibitor protein (GIP, causing suppression of host defense responses) and a necrosis-inducing protein 1 (NPP1).RESULTSThe translated ORF of P. cinnamomi GIP codifies a 269aa protein, with a predict Mw of 28,8KDa. Scanning against protein search databases revealed that P. cinnamomi GIPare a serine protease, with a trypsin domain profile. A characteristic feature of Ser proteases is to have a catalytic triad charge relay system, with residues of H, D and S in thatorder along the sequence, essential for the proteolytic function.In Figure 1 are shown the multiple alignment of various sequences who showed great homology with P.cinnamomi GIP, including another GIPs of Phytophthora genus, and a serine protease and a trypsin protease from P. infestans. GIPs have in common the fact that none ofthem have an intact catalytic triad, like other serine proteases, although they share with them several stretches of amino acids and motifs that are highly conserved. Thus, in allPhytophthora GIPs, there are substitutions in residues of the catalytic triad: H-79A,S,T,I,M-79 (in P. cinnamomi: S-79), D-128N-128 (only in P. cinnamomi and P. sojaeGIP2, and Ser-217T-217, in all Phytophthora GIPs.Therefore, GIPs are proteolytically inactive, referred as serine protease homologs, and presumably function as host-enzyme inhibitors. It can be hypothesized that a major role for GIP is to suppress the release of glucan elicitors during Phytophthora sp. infections, thereby reducing theeffectiveness of the plant host’s surveillance system (Rose et al., 2002).ACKNOWLEDGEMENTS: The Project COMBATINTA/SP2.P11/02 Interreg IIIA – Cross-Border Cooperation Spain-Portugal, financed by The European Regional Development Fund, and the Project “Identification,characterization and role of molecular factors associated with the mechanisms of infection of Fagaceae species by Phytophthora cinnamomi” (PTDC/AGR-AAM/67628/2006) FCT, supported this work.REFERENCESLarkin, M., Blackshields, G., Brown, N., Chenna, R., McGettigan, P., McWilliam, H., Valentin, F., Wallace, I., Wilm, A., Lopez, R., Thompson, J., Gibson, T. & Higgins D., 2007. Bioinformatics, 23(21): 2947-2948.Michiels, A., Tucker, M., Van Den Ende, W. & Van Laere, A., 2003. Chromosomal Walking of Flanking Regions From Short Known Sequences in GC-Rich Plant Genomic DNA. Plant Molecular Biology Reporter, 21:295-302.Rose, J., Ham, K., Darvill, A. & Albersheim, P., 2002. Molecular cloning and characterization of glucanase inhibitor proteins: coevolution of a counterdefense mechanism by plant pathogens. Plant Cell, 14:1329-1345.FIGURE 1 – Multiple sequence alignment of GIP and GIP-like genes from Phytophthora sp.B0B0H5_PHYCIN – putative GIP from P. cinnamomi; Q945U0_9STRA – GIP1 from P.sojae; B1AC90_PHYIN – GIP1 from P.infestans; Q945T9_9STRA – GIP2 from P.sojae;B1AC87_PHYIN – GIP2 from P.infestans; Q945T8_9STRA - GIP2 from P.sojae; B1AC88_PHYIN – GIP3 from P.infestans; Q2M411_PHYIN – trypsin protease GIP-like;B1AC89_ PHYIN – GIP4 from P.infestans; B1AC86_ PHYIN – serine protease from P.infestans; Q2M412_PHYIN- trypsin protease GIP-like.FIGURE 2 – Multiple sequence alignment of TGase genes fromPhytophthora sp. B0B0Q5_PHYCI - P. cinnamomi transglutaminase elicitor; Q6Q475_9STRA – P. sojaetransglutaminase elicitor; Q01928_PHYME – P. megasperma glycoprotein elicitor; Q6XDM3_PHYIN - P. infestanstransglutaminase elicitor M81C; Q6XDM2_PHYIN - P. infestans transglutaminase elicitor M81D)P. cinnamomi transglutaminase is a protein with 533 aa with a predict Mw of 57,7KDa. Phytophthora sp.TGases are even more closely related amongst them than GIPs, asshown in Figure 2.P. cinnamomi necrosis-inducing protein1 has 256 aa with a predict Mw of 29,0KDa. Scanning against protein search databases (data not shown) revealed that sequenceswho showed greater homology with P. cinnamomi NPP1 were two NPP1 proteins of P. infestans (Q2M430 _PHYIN; Q2M429 _PHYIN). The phylogram showing the moreclosely related sequences are shown in Figure 3.FIGURE 3 – Phylogram of most closely related P.cinnamomi NPP1 protein. B0B0Q6_PHYCI - P. cinnamomi NPP1;Q8LKL0_9STRA – P. sojae necrosis-inducing-like protein; Q9AT28_PHYPR – P. parasiticanecrosis-inducing-like protein; Q2M430_PHYIN - P. infestans NPP-like protein;Q2M429_PHYIN - P. infestans NPP-like protein.Further studies, including plant-pathogen phenotypic interactions areneeded to understand how each individual factor can affectpathogenesis mechanisms of P. cinnamomi, and how this knowledgecan be used for control ink disease and other Phytophthora sp.diseases .

Identification and characterization of molecular factors associated with the Phytophthora cinnamomi infection mechanisms

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Identification and characterization of molecular factors associated with the Phytophthora cinnamomi infection mechanisms

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