Chitosan induces differential transcript usage of chitosanase 3 encoding gene (csn3) in the biocontrol fungus Pochonia chlamydosporia 123

Abstract

Background: Pochonia chlamydosporia is an endophytic fungus used for nematode biocontrol that employs its cellular and molecular machinery to degrade the nematode egg-shell. Chitosanases, among other enzymes, are involved in this process. In this study, we improve the genome sequence assembly of P. chlamydosporia 123, by utilizing long Pacific Biosciences (PacBio) sequence reads. Combining this improved genome assembly with previous RNA-seq data revealed alternative isoforms of a chitosanase in the presence of chitosan. This study could open new insights into understanding fungal resistance to chitosan and root-knot nematode (RKN) egg infection processes.

Results: The P. chlamydosporia 123 genome sequence assembly has been updated using long-read PacBio sequencing and now includes 12,810 predicted protein-coding genes. Compared with the previous assembly based on short reads, there are 701 newly annotated genes, and 69 previous genes are now split. Eight of the new genes were differentially expressed in fungus interactions with Meloidogyne javanica eggs or chitosan. A survey of the RNA-seq data revealed alternative splicing in the csn3 gene that encodes a chitosanase, with four putative splicing variants: csn3_v1, csn3_v2, csn3_v3 and csn3_v4. When P. chlamydosporia is treated with 0.1 mg·mL^{- 1} chitosan for 4 days, csn3 is expressed 10-fold compared with untreated controls. Furthermore, the relative abundances of each of the four transcripts are different in chitosan treatment compared with controls. In controls, the abundances of each transcript are nil, 32, 55, and 12% for isoforms csn3_v1, csn3_v2, csn3_v3 and csn3_v4 respectively. Conversely, in chitosan-treated P. chlamydosporia, the abundances are respectively 80, 15%, 2-3%, 2-3%. Since isoform csn3_v1 is expressed with chitosan only, the putatively encoded enzyme is probably induced and likely important for chitosan degradation.

Conclusions: Alternative splicing events have been discovered and described in the chitosanase 3 encoding gene from P. chlamydosporia 123. Gene csn3 takes part in RKN parasitism process and chitosan enhances its expression. The isoform csn3_v1 would be related to the degradation of this polymer in bulk form, while other isoforms may be related to the degradation of chitosan in the nematode egg-shell.