Effectors

The discovery of pathogen effectors that can "manipulate host cell structure and function, thereby facilitating infection (virulence factors or toxins) and/or triggering defence responses (avirulence factors or elicitors)" (Kamoun 2006) has led to intense interest and effector-focused research. Although initially identified slowly and laboriously by positional cloning (Orbach et al. 2000; Dodds et al. 2004; Ridout et al. 2006; Gout et al. 2006), advances in DNA sequencing and bioinformatic technologies now facilitate the prediction of suites of candidate effectors in entire phytopathogen genomes (Torto et al. 2003; Choi et al. 2010). Typically, criteria used to interrogate pathogen genomes include identifying sequences encoding small proteins which are under diversifying selection and which have an N-terminal signal peptide (Mueller et al. 2008; Dean et al. 2005; Kamper et al. 2006; Haas et al. 2009). These search strategies have also been applied to cDNA libraries and EST (Expressed Sequence Tags) databases enriched with sequences from the biotrophic phase of pathogenic biotrophs or from the host-pathogen interface. This approach has, for example, resulted in the identification of effectors AvrM, AvrP4 and AvrP123 from M. lini (Catanzariti et al. 2006).

Many important insights into effector biology have been acquired by studying the sequences of predicted effectors. Analysis of the sequences of known Phytophthora effectors has, for example, led to identification of a conserved N-terminal RXLR dEER motif in these proteins (RXLR-X5-21-dEER; where upper case letters represent consensus amino acids in 10 out of 11 sequences and lowercase letters represent consensus amino acids in more than half of the genes) (Rehmany et al. 2005; Birch et al. 2006). Using this motif as a search criterion, 150-600 candidate effectors have been identified in the genomes of Phytophthora sojae and Phytophthora ramorum, the exact number obtained depending on the stringencies of the search settings, such as whether or not the dEER motif is also included and if hidden Markov Model statistics are applied (Win et al. 2007; Tyler et al. 2006). Other motifs that have been identified by sequence comparisons of potential Phytophthora effectors include the W-, L- and Y-motifs in the C-terminal portions of some RXLR effectors (Jiang et al. 2008). The W-motif is required for the HR mediated by the interaction between the Rpi-blb1 resistance protein and ipiO effectors (Champouret et al. 2009) and both W- and Y-motifs are required for suppression of programmed cell death by Avr1b (Dou et al. 2008a).

Another family of Phytophthora effectors known as Crinklers was identified by analysis of ESTs encoding secreted sequences (Torto et al. 2003). Members of this gene family in P. infestans, P. sojae and P. ramorum cause necrosis in host plants and contain a conserved LXLFLAK motif at the N-terminus (Haas et al. 2009; Win et al. 2007). Similar analyses of EST sequences isolated from barley epidermal cells containing B. graminis powdery mildew haustoria identified a Y/F/WxC-motif in small secreted proteins encoded by this fungus (Godfrey et al. 2010). However, not all pathogen effectors have such readily recognisable motifs and, to date, no highly conserved motif has been associated with other fungal effector sequences [e.g. in flax rust (Rafiqi et al. 2010) and in poplar rust (Joly et al. 2010)]. In the majority of cases, the function and biological significance of the putative effectors identified by bioinformatics remain to be determined. To this end, several high-throughput techniques for functional screening of potential effectors have been developed, including screening for phenotypes arising from expression of proteins in host plants transformed by Agrobacterium (Torto et al. 2003; Oh et al. 2009).

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