This difference may be due to different receptors, cellular contexts or experimental conditions

frame translation of H. arabidopsidis genome which is reported to have 134 candidate RxLR effectors was used as a positive control. Second, an HMM profile of the RxLR domain was constructed by manually aligning the RxLR domains of the 53 RxLR effectors from Phytophthora species and H. parasitica. The resulting alignment was fed to the hmmbuild program to generate the HMM profile. The HMM profile was used to search the translations for candidate effectors using the hmmsearch program. To validate our computational approach, the same HMM profile was used to search the six frame translation of H. arabidopsidis genome. Furthermore, the whole Pythium proteome was also searched with the HMM. Third, a comprehensive database of RxLR effector proteins from Phytophthora species, H. arabidopsidis, and A. laibachii was created. Putative homologs in predicted proteomes of Pythium were identified by BLASTP search against the RxLR effector database at E-value cutoff of 1025. Fourth, a string searches was performed for the RxLR, RxLx and Rx motif within the amino terminus of each six frame translation of Pythium genomes, 30 to 150 residues from the signal peptide. We computationally screened the six Pythium genomes for candidate YxSL effectors using a HMM profile of the putative YxSL motif, a novel effector motif identified first in Py. ultimum var. ultimum, constructed using 57 genes containing YxSL motif from three Phytophthora species and Aphanomyces eutieches. Using the YxSL motifs from Py. ultimum var. ultimum as a control, we identified an initial 23428871 set of 123 proteins containing the YxSL motif in 7 Pythium species. Using the same HMM profile we were able to identify 21 GSK126 price additional proteins with the YxSL motif in the Ph. infestans and Ph. sojae genomes. After searching against the HMM profile and multiple alignment of the proteins we selected a set of 141 proteins, which includes 120 candidate effectors from seven Pythium species and 21 from two Phytophthora species, with YxSL motif situated between 30 to 150 amino acids positions. For the CRN effectors, a BLASTP search against 21 welldefined amino-terminal domains from Ph. infestans and Py. ultimum var. ultimum CRN sequences was performed to identify proteins with putative LFLAK-like domains. The candidate CRN sequences from Ph. infestans and Pythium species were used to construct an HMM profile and the CRN sequences from Py. ultimum var. ultimum were used as 6099352 a control. Two criteria were used to identify candidate LxLYLAR/K proteins. First, the conserved motif should be preceded by a signal peptide and followed by WL motif. Second, the conserved motif should be located between 40 to 65 amino acids after first methionine. Using the HMM profile, we identified additional candidate effectors with an LxLYLAR/K domain. To validate our computational methods, the same HMM profile was used to identify the CRN effectors from H. arabidopsidis genome which is reported to have 20 candidate CRN effectors. Multiple alignments were conducted using the programs ClustalW and ClustalX. Sequence alignments were submitted to the WebLogo server to generate a sequence logo for the consensus. 10 mg/l FeSO47 H2O, 1 g/l yeast extract) at 25uC with shaking. Approximately 50 mg of hyphae growing out of the plugs were then transferred to flasks containing media for the various expression assays, with the exception of Py. iwayamai, mycelium was grown under the following conditions: 1, nutrientrich YEB medium for 3 days at 25uC frame translation of H. arabidopsidis genome which is reported to have 134 candidate RxLR effectors was used as a positive control. Second, an HMM profile of the RxLR domain was constructed by manually aligning the RxLR domains of the 53 RxLR effectors from Phytophthora species and H. parasitica. The resulting alignment was fed to the hmmbuild program to generate the HMM profile. The HMM profile was used to search the translations for candidate effectors using the hmmsearch program. To validate our computational approach, the same HMM profile was used to search the six frame translation of H. arabidopsidis genome. Furthermore, the whole Pythium proteome was also searched with the HMM. Third, a comprehensive database of RxLR effector proteins from Phytophthora species, H. arabidopsidis, and A. laibachii was created. Putative homologs in predicted proteomes of Pythium were identified by BLASTP search against the RxLR effector database at E-value cutoff of 1025. Fourth, a string searches was performed for the RxLR, RxLx and Rx motif within the amino terminus of each six frame translation of Pythium genomes, 30 to 150 residues from the signal peptide. We computationally screened the six Pythium genomes for candidate YxSL effectors using a HMM profile of the putative YxSL motif, a novel effector motif identified first in Py. ultimum var. ultimum, constructed using 57 genes containing YxSL motif from three Phytophthora species and Aphanomyces eutieches. Using the YxSL motifs from Py. ultimum var. ultimum as a control, we identified an initial set of 123 proteins containing the YxSL motif in 7 Pythium species. Using the same HMM profile we were able to identify 21 additional proteins with the YxSL motif in the Ph. infestans and Ph. sojae genomes. After searching against the HMM profile and multiple alignment of the proteins we selected a set of 141 proteins, which includes 120 candidate effectors from seven Pythium species and 21 from two Phytophthora species, with YxSL motif situated between 30 to 150 amino acids positions. For the CRN effectors, a BLASTP search against 21 welldefined amino-terminal domains from Ph. infestans and Py. ultimum var. ultimum CRN sequences was performed to identify proteins with putative LFLAK-like domains. The 12931192 candidate CRN sequences from Ph. infestans and Pythium species were used to construct an HMM profile and the CRN sequences from Py. ultimum var. ultimum were used as a control. Two criteria were used to identify candidate LxLYLAR/K proteins. First, the conserved motif should be preceded by a signal peptide and followed by WL motif. Second, the conserved motif should be located between 40 to 65 amino acids after first methionine. Using the HMM profile, 26013995 we identified additional candidate effectors with an LxLYLAR/K domain. To validate our computational methods, the same HMM profile was used to identify the CRN effectors from H. arabidopsidis genome which is reported to have 20 candidate CRN effectors. Multiple alignments were conducted using the programs ClustalW and ClustalX. Sequence alignments were submitted to the WebLogo server to generate a sequence logo for the consensus. 10 mg/l FeSO47 H2O, 1 g/l yeast extract) at 25uC with shaking. Approximately 50 mg of hyphae growing out of the plugs were then transferred to flasks containing media for the various expression assays, with the exception of Py. iwayamai, mycelium was grown under the following conditions: 1, nutrientrich YEB medium for 3 days at 25uC