No transmembrane-spanning region was identified using the TMHMM program. An extracellular localization was predicted by Neural nets using the ProComp program, suggesting that the encoded protein may be secreted. find more Cas3 and Cas4 share 98 % identity (100 % positive amino acids) with each other, with only one substitution at position 15 in the signal peptide. They share respectively 93 % and 94 % identity (98 % positive amino acids) with the reference Cas1 sequence. The predicted mature cassiicolin domain shows one positive substitution (S instead of T) compared to the reference Cas1 sequence. Cas2
remains the most divergent protein isoform with seven substitutions and one insertion relative to Cas1, as described previously (Déon et al. 2012). Fig. 1 Neighbor-joining phylogenetic tree of the cassiicolin precursor genes from four endophytic (E70, E78, E79 and E139) and two pathogenic strains of C. casiicolin (CCP and CC004). Bootstrap values are shown above the branch Fig. 2 The amino
acids sequence alignment of the cassiicolin precursor proteins Cas1 (ABV25895), Cas2 (ADC54229), Cas3 (AFH88923 and AFH88924) and Cas4 (AFH88925 and AFH88926). The mature cassiicolin domain is indicated by bold letters. The signal peptide is underlined. CLUSTAL W annotation: conserved amino acids (*); amino acids of strongly similar properties (:); amino acids of weakly similar properties (.) The 5′ and 3′ untranslated regions as well as the introns were the more divergent regions in the cas gene sequences. The ratio between the non-synonymous (d N ) and synonymous (d S ) substitution GDC 0449 rates was calculated for each sequence pair to estimate the selection pressure acting on the cas gene. This ratio could not be calculated among the C. cassiicola endophytes since a single divergent nucleotide only was this website observed in their coding region. The d N /d S ratios calculated between the
cas gene sequences from the isolates CCP, CC004 and the endophytes were all <1 (between Cediranib (AZD2171) 0.13 and 0.34) suggesting that the Cas gene may be under purifying selection pressure. Pathogenicity of the C. cassiicola endophytes Inoculations on detached leaves were performed to investigate the potential pathogenicity of the four C. cassiicola endophytic isolates on the cultivars from which they were originally isolated (Fig. 3). The pathogenic strain CCP was used as a control on both cultivars. The water controls remained negative over the whole experiment. No necrosis was observed at 1 and 2 days post-inoculation (dpi) regardless of the isolate. At 5 dpi, only pinpoint necroses were visible on the leaves inoculated with the endophytic strains E78, E79 and E139 isolated from the RRIM600 cultivar. However, plants inoculated with the pathogenic isolate CCP had already developed disease symptoms at this time as lesion size had reached 445 mm².