Distribution and expression of elicitin genes in the interspecific hybrid oomycete Phytophthora alni

Abstract

Phytophthora alni subsp. alni, P. alni subsp. multiformis, and P. alni subsp. uniformis are responsible for alder disease in Europe. Class I and II elicitin gene patterns of P. alni subsp. alni, P. alni subsp. multiformis, P. alni subsp. uniformis, and the phylogenetically close species P. cambivora and P. fragariae were studied through mRNA sequencing and 3' untranslated region (3'UTR)-specific PCRs and sequencing. The occurrence of multiple 3'UTR sequences in association with identical elicitin-encoding sequences in P. alni subsp. alni indicated duplication/recombination events. The mRNA pattern displayed by P. alni subsp. alni demonstrated that elicitin genes from all the parental genomes are actually expressed in this allopolyploid taxon. The complementary elicitin patterns resolved confirmed the possible involvement of P. alni subsp. multiformis and P. alni subsp. uniformis in the genesis of the hybrid species P. alni subsp. alni. The occurrence of multiple and common elicitin gene sequences throughout P. cambivora, P. fragariae, and P. alni sensu lato, not observed in other Phytophthora species, suggests that duplication of these genes occurred before the radiation of these species.

FIG. 1.

Multiple-amino-acid sequence alignment of the elicitins characterized in this study for P. alni, P. cambivora, and P. fragariae (AE1.1, AE1.2, AE2, BE1, BE2, and HAE1) and well documented acidic (α), basic (β), and highly acidic (HAE) elicitins retrieved from the GenBank database: P. cryptogea Z34462, Z34459, Z34460, Z34461 (24); P. infestans AY830090 (17); P. cinnamomi AJ000071 (11); P. sojae AJ007859 (1); and P. parasitica S67432 (6). The sequence corresponding to the signal peptide is underlined. pI are indicated in parentheses, following sequence references.

FIG. 2.

Phylogenetic relationships between elicitins, inferred from the sequence alignment of the mature polypeptides. The unrooted phylogram was constructed using a parsimony analysis and the neighbor-joining method, based on the multiple alignment of elicitin sequences listed in Fig. 1 and using the P. infestans inf6 sequence as an outgroup. Bootstrap values (>50%) from 10,000 replicates are indicated. AE1.1/0.2 and AE2 fall into the class I acidic elicitin group (solid line), whereas BE1 and BE2 are class I basic elicitins (dotted line). HAE1 is closely related to class II highly acidic elicitins (dashed line).

FIG. 3.

Sequence alignment of the regions corresponding to the 3′UTRs of the mRNAs, deduced from cDNA sequencing. 3′UTR-specific groups are indicated on the left and correspond to the clustering of identical or nearly identical sequences. Fourteen different 3′UTR groups were defined among the 28 sequences obtained from our 15-isolate panel. The different 3′UTR groups were designated according to the elicitin class, i.e., a for acidic, ha for highly acidic, and b for basic elicitins. For each 3′UTR, the boxed sequence represents the polymorphic region from which a 3′UTR-specific primer could be designed. The PFF309b 3′UTR sequence was so divergent that it could not be properly aligned. PFF309b was therefore not represented, but a specific reverse primer could be designed (a11-R).

FIG. 4.

Distribution of the acidic, basic, and highly acidic elicitin genes resolved in this study for P. alni subsp. alni (Paa), P. alni subsp. multiformis (Pam), P. alni subsp. uniformis (Pau), and the phylogenetically close species P. cambivora and P. fragariae sensu lato, as inferred from sequencing of cDNA and 3′UTR-specific PCR tests conducted on genomic DNA and cDNA libraries. Coding sequences are boxed and were found to be associated with different 3′UTRs. The occurrence of a1, a6, and a9 3′UTRs is deduced only from sequencing, as no 3′UTR-specific primer could be designed from these sequences. With the exception of the HAE1 associated with the ha1 3′UTR in P. cambivora, all the elicitin genes we resolved were shown to be expressed in our 15-isolate panel by 3′UTR-specific PCR with cDNA libraries. ^a, only for P. cambivora isolate PCjc17; ^Pff, obtained for P. fragariae var. fragariae; ^Pfr, obtained for P. fragariae var. rubi.