Selection of reference genes in Hedysarum coronarium under various stresses and stages of development.

The cultivation of Hedysarum coronarium has generated interest recently for its high yield as a fodder crop, its high protein content, and the presence of condensed tannins in its leaf and stem tissues. Gene expression studies can lead to a better understanding of the biological processes of live organisms. Specifically, reverse transcription followed by quantitative polymerase chain reaction (PCR) represents the most powerful technology for comparing the expression profiles of target genes. The use of reference genes as internal controls to normalize messenger RNA (mRNA) levels is a requirement of quantitative PCR (qPCR). Few studies on reference genes have been performed in plants, and no studies have been performed in H. coronarium. Therefore, the aim of this study was to identify and evaluate reference genes to use in qPCR in H. coronarium. Sulla tissues under two conditions of abiotic stress and at various stages of development were studied to determine adequate reference genes. To optimize the identity and number of reference genes, geNorm and BestKeeper software programs were employed. Based on the results of both analyses, TUA1, TUA2, and UBQ were found to be the most suitable reference genes, and the combination of these three genes was suggested for the accurate normalization of gene expression in sulla tissues.

Selection of housekeeping genes for normalization by real-time RT-PCR: analysis of Or-MYB1 gene expression in Orobanche ramosa development.

Real-time PCR has become the method of choice for accurate and in-depth expression studies of candidate genes. To avoid bias, real-time PCR is referred to one or several internal control genes that should not fluctuate among treatments. A need for reference genes in the parasitic plant Orobanche ramosa has emerged, and the studies in this area have not yet been evaluated. In this study, the genes 18S rRNA, Or-act1, Or-tub1, and Or-ubq1 were compared in terms of expression stability using the BestKeeper software program. Among the four common endogenous control genes, Or-act1 and Or-ubq1 were the most stable in O. ramosa samples. In parallel, a study was carried out studying the expression of the transcription factor Or-MYB1 that seemed to be implicated during preinfection stages. The normalization strategy presented here is a prerequisite to accurate real-time PCR expression profiling that, among other things, opens up the possibility of studying messenger RNA levels of low-copy-number-like transcription factors.

First Report of Orobanche crenata on Sulla (Hedysarum coronarium) in Andalusia, Southern Spain.

Crenata broomrape (Orobanche crenata Forsk.), an obligate chlorophyll-lacking root parasite, is the major constraint for growing legume crops in infested soils in southern Spain. Peas (Pisum sativum L), faba beans (Vicia faba L.), and narbon bean (Vicia narbonensis L.) are seriously affected (1,2,4). However, no information is available regarding its ability to attack sulla (Hedysarum coronarium L.), which is an important fodder legume grown in rain-fed conditions, yielding as much as 10,000 kg/ha of dry matter. In 2008, we found broomrape shoots infecting 4 to 5% of sulla plants in a trial field (two land races, P-26 and P-3, in a 500-m2 area) in Cordoba (Andalusia, southern Spain). The shoots developed numerous fertile flowering stalks throughout the field. The previous crop, narbon bean, was heavily infected during 2007 by O. crenata. Infection of sulla plants was confirmed by digging up the plant to verify the attachment of the broomrape plant to the sulla roots. The level of infection was low with only one to two emerged broomrape shoots per sulla plant infested. Morphology was typical of O. crenata. The calyx of the flowers was 13 to 18 mm long with free, bidentate segments. The white corolla of the flowers was 18 to 28 mm long and glandular pubescent. The lips were divergent, large, not ciliate, and filaments inserted 2 to 3 mm above the base of the corolla are hairy at base with glandular hair at the apex. The anthers were brown (3). Specimens were deposited in the Herbarium of the University of Córdoba (identification number COA 45358). To our knowledge, this first report of O. crenata parasitizing sulla shows that control methods for O. crenata will be needed if sulla is to be used as an alternative legume fodder in the infested soils of southern Spain. References: (1) J. I. Cubero and M. T. Moreno. Page 41 in: Some Current Research on Vicia faba in Western Europe. D. A. Bond et al., eds. Luxembourg, 1979. (2) S. Nadal et al. Plant Breed. 126:110, 2007. (3) A. J. Pujadas-Salvá. Page 187 in: Resistance to Orobanche: The State of the Art. Junta de Andalucía, Sevilla, Spain, 1999. (4) D. Rubiales et al. Crop Prot. 22:865, 2003.

Role of cell wall-degrading enzymes in pathogenicity of Fusarium oxysporum.

Fusarium oxysporum invades its host plants through the roots and colonizes the vascular system. It produces a great variety of cell-wall degrading enzymes (CWDE), such as cellulases, xylanases, pectinases and proteases. Our group has purified and characterized an endopolygalacturonase (PG1), two exopolygalacturonases (PG2 and PG3), an endoxylanase (XYL1) and an endo pectatelyase (PL1). We have isolated the following CWDE-encoding genes: pg1, pgx4, pg5, xyl2, xyl3, prt1 and pl1. Gene expression in different culture conditions has been determined by Northern analysis. The occurrence of these genes in different formae speciales has been analyzed by Southern analysis and PCR. All these genes are expressed during different stages of the interaction with the host plant indicating a possible role in pathogenesis. At present, targeted gene disruption is being carried out, in order to determine the role of each gene in the pathogenicity process.