IDENTIFICATION AND QUANTIFICATION OF DIFFERENTIALLY EXPRESSED GENES ASSOCIATED WITH CITRUS BLIGHT (Citrus spp.)

José Renato De Abreu,Ariadne Ribeiro Henriques,Antonio Chalfun-Junior,Anderson Tadeu Silva,Miguel Angel Dita Rodríguez,Luciano Vilela Paiva

doi:10.1590/s1413-70542015000100004

Abstract

Brazil is the largest citrus producer in the world, being responsible for more than 20% of its production, which is, however still low due to phytosanitary issues such as citrus blight. Citrus blight is an anomaly whose causes still have not yet been determined, therefore there are no efficient control measures to minimize the production losses with the use of resistant varieties being considered the most appropriate method. However, little is known about the genes involved in the defense response of the plants to this anomaly. Considering that many physiological alterations associated with plant stress responses are controlled at a transcriptional level, in this study we sought the identification and characterization of the gene expression products differentially expressed in the response to the citrus blight. Through the suppressive subtractive hybridization technique, expressed cDNA libraries were built using mRNAs isolated from "Cravo" lemon tree roots (Citrus limonia L. Osbeck) under "Pera" orange (Citrus sinensis L. Osbeck) of healthy and sick plants. 129 clones were obtained by subtraction and their sequences were compared in databases. 34 of them linked to proteins associated to stress processes, while the others were similar to sequences of unknown functions or did not present similarity with sequences deposited in the databases. 3 genes were selected and their expressions were studied by RT - qPCR in real-time. Plants with citrus blight presented an increase of the expression level in two of those genes, suggesting that these can be directly involved with this anomaly.

Highlights

The citrus blight is an alteration in the normal development of the plant, characterized by accentuated loss of leaves, excess branches and shoots on the trunk, gradual drying of branches, unseasonal flowering, accentuated nutrient deficiencies, accumulation of soluble phenols in the stem, presence of amorphous and/ or filamentous obstructions in the xylem vessels and low production
That implicates in the inexistence of prophylactic measures for the control of the citrus blight, so that the only recommended practice is the removal of the sick plants when they enter into an economically unviable production phase
Two subtractive libraries were built, one enriched with cDNAs differentially expressed in sick plants and another enriched with cDNAs differentially expressed in healthy plants. 38 clones of the forward library and 91 of the reverse library were obtained (Table 1 and 2), that were identified, by database comparison, as sequences associated to stress processes

Summary

Introduction

The citrus blight is an alteration in the normal development of the plant, characterized by accentuated loss of leaves, excess branches and shoots on the trunk, gradual drying of branches, unseasonal flowering, accentuated nutrient deficiencies, accumulation of soluble phenols in the stem, presence of amorphous and/ or filamentous obstructions in the xylem vessels and low production The affected plants rarely die, but they become economically unviable due to low amount and quality of the produced fruits. This anomaly has been known for more than a century, the causal agent is still not known (Barrios et al, 2006; Brlansky et al, 2012). That implicates in the inexistence of prophylactic measures for the control of the citrus blight, so that the only recommended practice is the removal of the sick plants when they enter into an economically unviable production phase

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