Original article
Identification of sugarcane genes induced in disease-resistant somaclones upon inoculation with Ustilago scitaminea or Bipolaris sacchari

https://doi.org/10.1016/j.plaphy.2005.07.011Get rights and content

Abstract

To understand the molecular basis of a specific plant-pathogen interaction, it is important to identify plant genes that respond to the pathogen attack. Amplified fragment length polymorphism (AFLP) analysis of cDNA was used to identify sugarcane genes differentially expressed in disease-resistant but not in susceptible sugarcane somaclones in response to inoculation with either Ustilago scitaminea or Bipolaris sacchari (also known as Helminthosporium sacchari or Drechslera sacchari), causal agents of smut and eyespot respectively. In total 62 differentially regulated genes were identified, of which 10 were down-regulated and 52 were induced. Of these 52, 19 transcript derived fragments showed homology to known plant gene sequences, most of them related to defense or signaling. The total set of differentially expressed sugarcane genes can be an important resource for further studies aimed at understanding sugarcane pathogen defense.

Introduction

Sugarcane (Saccharum spp.) is a tropical grass that originates from Asia where it has been cultivated for over 4,000 years. Over 2,000 years ago methods for extracting sugar from sugarcane were developed in India. Nowadays, sugarcane is cultivated in tropical and sub-tropical regions throughout the world and over 60% of the world's sugar is extracted from sugarcane [15], [17]. In Cuba, sugarcane is one of the most important cultivated crops. However, fungal diseases such as eyespot and smut, caused by Bipolaris sacchari (E.J. Butler) Shoemaker (also known as Helminthosporium sacchari or Drechslera sacchari) and Ustilago scitaminea Syd. & P. Syd., respectively, can affect the agronomic performance of this crop in sugarcane-growing areas and cause losses as high as 40% of the total yield [10], [15].

B. sacchari causes typical eyespot symptoms; reddish elliptical lesions surrounded by a yellowish halo (Fig. 1B). These lesions develop into streaks and eventually the entire leaf may become necrotic. Eyespot can cause heavy losses if conditions favor disease development. However, nowadays, mostly resistant varieties are grown [15].

In contrast to B. sacchari, the causal agent of eyespot, U. scitaminea, the causal agent of smut, also affects the stalk of the cane. This disease can quickly devastate susceptible varieties dependent on the environmental conditions. The most predominant symptom is the occurrence of a so-called smut-whip composed of fungal and plant tissues emerging from the top of the plant [15] (Fig. 1 A).

Little is known about the molecular background of the interaction between pathogen and host in both pathosystems. This is largely due to the poor availability of genetics tools for Saccharum spp. in general. Only a few reports have been published describing some aspects of the molecular basis of the interaction. For sugarcane smut disease it has been demonstrated that the level of different polyamines and possible conjugation to phenolics in mature organs of U. scitaminea-infested and non-infested sugarcane plants correlates with smut susceptibility, suggesting that polyamine conjugation to phenolics might act as a mechanism of resistance or defence against this disease [23], [31].

A recent report describes the use of cDNA-AFLP and suppression subtractive hybridization (SSH) to identify differentially expressed sugarcane genes upon inoculation with the sugarcane smut fungus U. scitaminea. Using a restriction fragment length polymorphism (RFLP) approach, markers were developed and used on a population of 78 well characterized sugarcane genotypes that are used in a breeding program. In this study, 59 polymorphisms showing correlation with smut resistance were identified [7].

For B. sacchari, a study has been published describing resistance evaluations to eyespot of four somaclones under field and laboratory conditions. The analysis of the mitochondrial genome of the resistant somaclones provided evidence of somaclonal variation at the molecular level. It was suggested that specific changes in DNA patterns, including the appearance and disappearance of bands, appeared to be characteristic for the resistant genotypes [21].

Somaclonal variation has been widely employed for plant genetic improvement [19]. In sugarcane, this source of variability has been used for the selection of genotypes that are resistant to certain biotic and abiotic stress stimuli [8], [22]. Through this approach, somaclonal variants of sugarcane have been obtained that display a high degree of resistance to eyespot disease under laboratory and field conditions [8], [21]. Likewise, clones obtained by somaclonal variation and mutation have been identified displaying resistance to smut and other diseases. These clones have shown stable resistance now for more than 10 years [3].

To gain more insight in the genetic background of the enhanced resistance that is displayed by somaclonal sugarcane variants, one approach could be to perform global analysis of gene expression. Assuming that a gene is usually transcribed only when and where its function is required, determining the conditions under which a gene is expressed allows making inferences about its function [34]. The aim of this work is to identify genes that are activated and associated with resistance in sugarcane upon inoculation with either U. scitaminea or B. sacchari. This is pursued by isolating differentially expressed transcription derived fragments (TDFs) that are induced upon fungal challenge of resistant and susceptible sugarcane somaclones based on a cDNA-AFLP approach.

Section snippets

Results

Sugarcane plants were artifically infected under field conditions with either of the pathogens U. scitaminea or B. sacchari, respectively. For both pathogens the development of the infection on susceptible genotypes largely depends on environmental conditions that can hardly be controlled under field conditions. Therefore, symptom development was regularly monitored to determine the appropriate time points for leaf material to be collected. For B. sacchari clear symptoms were visible one week

Discussion

Due to the limited availability of genomic tools for Saccharum spp. only few studies have been conducted providing insight in the molecular background of pathogen resistance. In order to identify genes that possibly contribute to the establishment of disease resistance in somaclones that are resistant to either sugarcane smut, caused by U. scitaminea, or eyespot, caused by B. sacchari, cDNA-AFLP analysis was performed. On one hand, knowledge of the expression patterns of specific genes can

Inoculation of leaf material and sample collection

The disease-resistant somaclonal sugarcane variants used in this study are Ja S-44 (resistant to U. scitaminea) and CC 54-84 (resistant to B. sacchari) [3], [21]. In addition, the Ja 60-5 (susceptible to U. scitaminea) and C 87-51 (susceptible to B. sacchari) parental lines were used as controls. Twenty plants from Ja S-44 and the corresponding donor variety (Ja 60-5) were inoculated with teliospores of U. scitaminea, isolated from whips collected from Saccharum hybrid, var. Barbados 42231

Acknowledgements

B.P.H.J.T. is supported by a VENI grant of the Research Council for Earth and Life sciences (ALW) of the Netherlands Organization for Scientific Research (NWO).

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