Defence transcriptome assembly and pathogenesis related gene family analysis in Pinus tecunumanii (low elevation)

Abstract

Background: Fusarium circinatum is a pressing threat to the cultivation of many economically important pine tree species. Efforts to develop effective disease management strategies can be aided by investigating the molecular mechanisms involved in the host-pathogen interaction between F. circinatum and pine species. Pinus tecunumanii and Pinus patula are two closely related tropical pine species that differ widely in their resistance to F. circinatum challenge, being resistant and susceptible respectively, providing the potential for a useful pathosystem to investigate the molecular responses underlying resistance to F. circinatum. However, no genomic resources are available for P. tecunumanii. Pathogenesis-related proteins are classes of proteins that play important roles in plant-microbe interactions, e.g. chitinases; proteins that break down the major structural component of fungal cell walls. Generating a reference sequence for P. tecunumanii and characterizing pathogenesis related gene families in these two pine species is an important step towards unravelling the pine-F. circinatum interaction.

Results: Eight reference based and 12 de novo assembled transcriptomes were produced, for juvenile shoot tissue from both species. EvidentialGene pipeline redundancy reduction, expression filtering, protein clustering and taxonomic filtering produced a 50 Mb shoot transcriptome consisting of 28,621 contigs for P. tecunumanii and a 72 Mb shoot transcriptome consisting of 52,735 contigs for P. patula. Predicted protein sequences encoded by the assembled transcriptomes were clustered with reference proteomes from 92 other species to identify pathogenesis related gene families in P. patula, P. tecunumanii and other pine species.

Conclusions: The P. tecunumanii transcriptome is the first gene catalogue for the species, representing an important resource for studying resistance to the pitch canker pathogen, F. circinatum. This study also constitutes, to our knowledge, the largest index of gymnosperm PR-genes to date.

Keywords: Disease resistance; Fusarium circinatum; PR genes; Pinus patula; Pinus tecunumanii; Transcriptome assembly.

Fig. 1

Fusarium circinatum disease progression on inoculated low elevation Pinus tecunumanii and Pinus patula seedlings. Error bars represent the standard error of the mean (Inoc n = 100; Mock n = 20). Pnte – P. tecunumanii; Pipt – P. patula; Inoc – inoculated; Mock – Mock-inoculated; * - significant difference between inoculated and mock-inoculated groups (Kruskal-Wallis rank sum test, p < 0.05)

Fig. 2

Summarised statistics for the longest isoforms per gene for preliminary assemblies. Left – Pinus tecunumanii (LE) assemblies (Pnte). Right – Pinus patula assemblies (Pipt). a Size, length, predicted open reading frame (ORF) content and longest transcript per assembly statistics. b N50, mean length and %GC. The grey, dashed, secondary y-axes only applies to the dashed grey lines. The x-axes represent respective assemblies. In each case the first four assemblies were produced using the full dataset and the remaining 16 with the normalised dataset. Numbers in assembly identities (25 & 31) represent the k-mer size used, ‘c’ indicates usage of CuffFly transcript reconstruction algorithm, ‘g’ indicates genome guided assemblies, ‘n’ indicates usage of the normalised dataset, ‘HS’ and ‘LS’ indicate usage of high- and low-stringency parameters respectively

Fig. 3

Distribution of biological process and molecular function Gene Ontology (GO) terms in the assembled transcriptomes. Green – Pinus tecunumanii assembly. Blue – Pinus patula assembly

Fig. 4

Comparison of completeness, contiguity and redundancy for assembled transcriptomes (underlined) to other available gymnosperm assemblies. BUSCOs were identified for the (a) eukaryotic (n = 303) and (b) embryophyta (n = 1440) lineage datasets. The primary y-axis refers to the percentage of BUSCOs in each category. The secondary, dashed, y-axis refers to the total contig count in each assembly

Fig. 5

Comparison of orthogroup size across lineages between two chitinase orthogroups. a PR-3 orthogroups OG0000134 (brown) and OG000642 (purple). b PR-8 orthogroups OG0000252 (brown) and OG0000455 (purple). In both cases the x-axis refers to the amount of proteins per species present in each orthogroup

Fig. 6

Putative Pathogenesis Related orthogroup transcript counts in P. tecunumanii and P. patula. The y-axis represents the transcript count for each orthogroup