Pathogens are able to deliver small-secreted, cysteine-rich proteins into plant cells to enable infection. The computational prediction of effector proteins remains one of the most challenging areas in the study of plant fungi interactions. At present, there are several bioinformatic programs that can help in the identification of these proteins; however, in most cases, these programs are managed independently. Here, we present EffHunter, an easy and fast bioinformatics tool for the identification of effectors. This predictor was used to identify putative effectors in 88 proteomes using characteristics such as size, cysteine residue content, secretion signal and transmembrane domains.

Black Sigatoka is a disease that occurs in banana plantations worldwide. This disease is caused by the hemibiotrophic fungus Pseudocercospora fijiensis, whose infection results in a significant reduction in both product quality and yield. Therefore, detection and identification in the early stages of this pathogen in plants could help minimize losses, as well as prevent the spread of the disease to neighboring cultures. To achieve this, a highly sensitive SPR immunosensor was developed to detect P. fijiensis in real samples of leaf extracts in early stages of the disease. A polyclonal antibody (anti-HF1), produced against HF1 (cell wall protein of P. fijiensis) was covalently immobilized on a gold-coated chip via a mixed self-assembled monolayer (SAM) of alkanethiols using the EDC/NHS method. The analytical parameters of the biosensor were established, obtaining a limit of detection of 11.7 µg mL−1, a sensitivity of 0.0021 units of reflectance per ng mL−1 and a linear response range for the antigen from 39.1 to 122 µg mL−1. No matrix effects were observed during the measurements of real leaf banana extracts by the immunosensor. To the best of our knowledge, this is the first research into the development of an SPR biosensor for the detection of P. fijiensis, which demonstrates its potential as an alternative analytical tool for in-field monitoring of black Sigatoka disease.

Substantial progress in the understanding of the events that govern embryo formation has been achieved in a variety of animal and nonplant systems; however, much less is known of this subject in plants (Brownlee and Berger, 1995). During embryogenesis a precise control of events such as cell division, differentiation, and growth is required (Turner, 1991). In animal systems these events appear to be regulated by protein phosphorylation through a concerted action of kinases (Simon et al., 1993;Turner, 1994; Hou et al., 1995) and phosphatases (Cyert and Thorner, 1989;Sun and Tonks, 1994). Most of the protein phosphorylation in cells occurs in residues of Ser and Thr, and in minor proportion in residues of Tyr. In the case of Tyr phosphorylation, a role in embryogenesis in animal cells has been suggested, since changes in the levels of protein Tyr phosphorylation have been shown to accompany embryo development in vertebrates (Maher and Pasquale, 1988;Turner, 1991Turner, , 1994 Gilardi-Hebenstreit et al., 1992; Nieto et al., 1992;Snider, 1994) and invertebrates (Shilo, 1992;Perrimon, 1993).The occurrence of protein phosphorylation and kinase activity in plants has been reported in several species (for review, see Stone and Walker, 1995). According to these reports, it seems that protein phosphorylation occurs more abundantly in Ser and Thr residues than in Tyr residues (Reddy et al., 1987;Saluja et al., 1987;Stone and Walker, 1995). Nevertheless, Tyr protein phosphorylation and Tyr kinase activity have already been reported in a few plant species, such as pea (Torruela et al., 1986; Håkansson and Allen, 1995), alfalfa (Duerr et al., 1993), tobacco (Suzuki and Shinshi, 1995;Zhang et al., 1996), and maize (Trojanek et al., 1996).The present study reports the occurrence of protein kinase activities in developing coconut zygotic embryos, which can phosphorylate proteins in Thr, Ser, and Tyr residues. Particular changes in the patterns of phosphorylated proteins and Tyr kinase activity during coconut embryo development are also described. MATERIALS AND METHODS Plant MaterialSeeds were collected from coconut (Cocos nucifera L.) groves in the Yucatán Península, México, and transported to the laboratory. The embryo developmental stages were arbitrarily defined, taking the first pollinated inflorescence as stage 0; stages 1 to 16 correspond to inflorescences of increasing maturity. Embryos were excised from the seeds in the laboratory and immediately processed. Tissue HomogenizationEmbryos were excised from seeds and immediately homogenized in buffer containing: 50 mm Tris-HCl, pH 7.4, 10 mm sodium pyrophosphate, 50 mm NaCl, 250 mm Suc, 10% glycerol, 1 mm EGTA, 0.2 mm orthovanadate, 1 mm 1 This work was supported by Fogarty International Research Collaboration Award (grant no. RO3TW00263), the Consejo Nacional de Ciencia y Tecnología (CONACYT) (grant no. 0014P-N9506), the Commission of the European Communities EC-STD (grant no. ERBTS3*CT940298), and a CONACYT Fellowship to I.I.-F. (no. 89535).* Corresponding author; e-...

High-quality RNA preparations are critical for further applications such as reverse transcriptase-polymerase chain reaction (RT-PCR) transcript amplifications, and elaboration of cDNA and expressed sequence tag libraries. Melanins are phenolic compounds present in many fungi and apparently play key roles in fungi pathogenesis and survival. However, during RNA extraction these compounds constitute a significant challenge to extraction of substantial quantities of high-quality RNA, and consequently to preparation of cDNA libraries. No method currently exists for RNA extraction from Mycosphaerella fijiensis that produces high quantities of melanin-free RNA. This fungus is the most important pathogen of cultivated Musa sp. varieties. A comparison is made between results obtained from the Trizol and RNeasy protocols for RNA extraction, two commercially available methods commonly used to obtain RNA from various sources. An improved methodology is described that allows isolation of intact RNA and elimination of melanins from M. fijiensis mycelium. RNA quality is evaluated by electrophoresis in formaldehyde-agarose gels, RT into cDNAs, and subsequent PCR amplification using primers designed against actin and beta- tubulin from fungi.

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