Differential early gene expression in Phaseolus vulgaris to Mexican isolates of Colletotrichum lindemuthianum in incompatible and compatible interactions q Sau ´l Fraire-Vela ´zquez a,b , Edmundo Lozoya-Gloria b, * a Unidad Acade ´mica de Biologı ´a Experimental, Universidad Auto ´noma de Zacatecas, C.P. 98600, Zacatecas, Zacatecas, Mexico b Irapuato Unit, CINVESTAV-IPN, Department of Genetic Engineering, P.O. Box 629, Libramiento Norte Carretera Irapuato-Leo ´n, C.P. 36500 Irapuato, Guanajuato, Mexico Accepted 22 October 2003 Abstract Plants of Phaseolus vulgaris cv. Michigan Dark Red Kidney and two Mexican pathotypes of Colletotrichum lindemuthianum showing contrasting interactions, compatible with race 2 and incompatible with race 1472, were analyzed by cytological and molecular methods. All conidia germinated 12 h post-inoculation. In the incompatible interaction the conidia were fragmented, their development arrested, few appressoria were observed and no hyphae were found. In contrast, in the compatible interaction the conidia developed typical appressoria and infective hyphae. The phenylalanine ammonia-lyase mRNA increased slightly after 4 h in the compatible interaction and more strongly after 2 h in the incompatible one. cDNA fragments of expressed genes sampled 0.5 – 6 h post-inoculation in incompatible interaction were cloned after subtractive hybridization. Preferentially expressed cDNAs were selected by reverse northern, confirmed by respective northern blots against total RNA, and assessed by image analysis. cDNA analysis of three early differentially expressed cDNAs matched b-glucosidase, Sina (Seven in absentia) and ubiquitin-like genes. The presumptive function of these genes in the plant defense response is discussed. q 2004 Elsevier Ltd. All rights reserved. Keywords: C3HC4-type RING zinc finger; Co-1; Colletotrichum lindemuthianum; Differential gene expression; Image analysis; Phaseolus vulgaris; Reverse northern; SMT3; Subtractive hybridization; SUMO 1. Introduction The interaction between French bean (Phaseolus vul- garis) and the fungus causing anthracnose (Colletotrichum lindemuthianum) has been studied widely. Cotyledons, excised hypocotyls or bean cell suspension cultures inoculated with conidia or in the presence of different elicitors have been tested [5,14,15,58,65]. Cell suspension cultures are suitable for biochemical and molecular studies because of their sensitivity to elicitors and rapid response of many genes to induction or suppression. However, they do not provide information about the cytology of the infection process especially on the timing and type of infection structures, or about the plant response to compatible and incompatible interactions. These aspects have been better studied by the use of excised hypocotyls and whole plants [41,50]. Phytoalexins in P. vulgaris are isoflavonoids arising from the phenylpropanoid biosynthetic pathway. This pathway is also responsible for lignin production [13,65]. Key initial enzymes are L-phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS). The corresponding transcripts and genes for these enzymes have been assayed and isolated, and transient increases in both interactions were regarded as establishment of a defense response [5,15,18,58]. Other factors related to the defense response include the increase in hydrolytic enzyme activity such as glucanases and chitinases [12,25,40], and the re-inforcement of structural barriers with lignin-like material and hydroxyproline-rich glycoproteins (HRGPs) [10,59]. In cell suspension cultures of P. vulgaris (cv. The Prince or cv. Canadian Wonder) 0885-5765/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2003.10.004 Physiological and Molecular Plant Pathology 63 (2003) 79–89 www.elsevier.com/locate/pmpp q The nucleotide sequences of the partial cDNAs isolated in this work have been submitted to GeneBank under accession numbers: AF451278 for ubiquitin-like gene; AF451279 for b-glucosidase gene and AF451280 for SINA gene. * Corresponding author. Tel.: þ52-462-623-96-59; fax: þ 52-462-624- 58-49. E-mail address: elozoya@ira.cinvestav.mx (E. Lozoya-Gloria).