IDENTIFICACIÓN DE PROTEÍNAS EFECTORAS MEDIANTE ESPECTROMETRÍA DE MASAS MALDI TOF/TOF DEL NEMATODO AGALLADOR DE LA RAÍZ Meloidogyne javanica

Abstract

<p><strong>Background:</strong> The main problem in grapevine cultivation is root-attachment nematodes cause serious yield problems in most crops worldwide. Through their different infection mechanisms these nematodes synthesize and secrete a mixture of protein-based effectors that they use to penetrate the root, migrate and develop into giant root feeding cells in host plants. The use of new molecular tools such as MALDI TOF/TOF (Matrix-Assisted Laser Desorption/Ionization - Time-Of-Flight) mass spectrometry and PCR (Polymerase Chain Reaction) technique have allowed us to know these proteins and genes in different microorganisms. <strong>Objective:</strong> To characterize the root-knot nematode <em>Meloidogyne javanica</em> by sequencing the 18S rRNA gene from infected root samples and the effector proteins of juvenile (J2) and adult (J4) stage of <em>M. javanica</em> by MALDI TOF/TOF shotgun proteomics dual mass spectrometry. <strong>Methodology:</strong> Infected roots of grapevine crop were collected to extract fresh galls and J2 of <em>M. javanica</em>, then inoculated on tomato plants. J4 of <em>M. javanica</em> were used for genomic DNA extraction and sequencing at the 18S rRNA gene level. The J2 and J4 stages of <em>M. javanica </em>were disinfected with sodium hypochlorite (0.5%) and sterile distilled water for protein extraction and analysis with MALDI-TOF/TOF. Finally, the sequences obtained were processed with ProteinPilot™ and Protein BLAST software for the identification of effector proteins of <em>M. javanica. </em><strong>Results:</strong> The nematode <em>M. javanica</em> was molecularly identified by PCR amplification of the 18S rDNA gene <em>M. javanica</em> with an identity percentage of 98% from infected root samples and by MALDI TOF/TOF mass spectrometry, effector protein sequences were identified such as: Beta-1,4-endoglucan and polygalaturonase, identified from J2, and expansin B2, CLAVATA3/ESR, Pectate lyase and Chorismato mutase from J4, involved in the different infection processes. In addition, we were able to identify 49 nematode non-effector proteins in both stages related to conserved biological development.<strong> Implications:</strong> The results indicate the existence of effector proteins related to root gill formation. <strong>Conclusions: </strong>This study confirms that dual mass spectrometry methodology provides in a rapid and reproducible way a proteomic profile that the galls nematode synthesizes to infect root cells and that can be used in other types of pathogens.</p>