Host-induced aneuploidy and phenotypic diversification in the Sudden Oak Death pathogen Phytophthora ramorum

Abstract

Background: Aneuploidy can result in significant phenotypic changes, which can sometimes be selectively advantageous. For example, aneuploidy confers resistance to antifungal drugs in human pathogenic fungi. Aneuploidy has also been observed in invasive fungal and oomycete plant pathogens in the field. Environments conducive to the generation of aneuploids, the underlying genetic mechanisms, and the contribution of aneuploidy to invasiveness are underexplored. We studied phenotypic diversification and associated genome changes in Phytophthora ramorum, a highly destructive oomycete pathogen with a wide host-range that causes Sudden Oak Death in western North America and Sudden Larch Death in the UK. Introduced populations of the pathogen are exclusively clonal. In California, oak (Quercus spp.) isolates obtained from trunk cankers frequently exhibit host-dependent, atypical phenotypes called non-wild type (nwt), apparently without any host-associated population differentiation. Based on a large survey of genotypes from different hosts, we previously hypothesized that the environment in oak cankers may be responsible for the observed phenotypic diversification in P. ramorum.

Results: We show that both normal wild type (wt) and nwt phenotypes were obtained when wt P. ramorum isolates from the foliar host California bay (Umbellularia californica) were re-isolated from cankers of artificially-inoculated canyon live oak (Q. chrysolepis). We also found comparable nwt phenotypes in P. ramorum isolates from a bark canker of Lawson cypress (Chamaecyparis lawsoniana) in the UK; previously nwt was not known to occur in this pathogen population. High-throughput sequencing-based analyses identified major genomic alterations including partial aneuploidy and copy-neutral loss of heterozygosity predominantly in nwt isolates. Chromosomal breakpoints were located at or near transposons.

Conclusion: This work demonstrates that major genome alterations of a pathogen can be induced by its host species. This is an undocumented type of plant-microbe interaction, and its contribution to pathogen evolution is yet to be investigated, but one of the potential collateral effects of nwt phenotypes may be host survival.

Keywords: Aneuploidy; Invasive pathogens; Loss of heterozygosity; Transposable elements.

Fig. 1

Nwt colony phenotype was experimentally reproducible by oak passage experiment. a Scraping off the outer bark 20 weeks post-inoculation revealed diseased tissue. The red mark at the center indicates the inoculation point. Isolates recovered from circle and square marks yielded wt and nwt colonies, respectively. b Isolates show diverse colony morphology. Top left: Pr-745 (wt, bay), top right: Pr-710 (wt, bay), both are wild type isolates used as inocula. Middle left and right: examples of re-isolates from canyon live oak showing nwt colony morphology. Bottom left: a re-isolate from canyon live oak showing a wt colony morphology, bottom right: an example of early senescence

Fig. 2

Transcriptome analyses support host-induced phenotypic diversification. a Eight cDNA samples from Petri plate cultures were clustered based on their global expression patterns of 14,339 transcripts. Group A consists of the two California bay isolates used as inocula and re-isolates from canyon live oak, all having wt colony phenotype. Isolates in group B are either from naturally infected coast live oak or from artificially-inoculated canyon live oak, and all have nwt colony phenotype. b Examples of mRNA profiles of P. ramorum genes differentially expressed between group A and group B. The order of isolates is same as that in a. A gene model number and its annotation are shown for each profile. Gene expression of PR_72359 and PR_49580 were estimated by qRT-PCR, and log2 fold changes (−ΔΔCT) are shown. PR_76099 was used as endogenous control gene, and expression levels were standardized to the genome sequence strain Pr-102. For PR_49580, because the reference PR-102 had a high expression level, its expression was offset by 12 for the presentation purpose. Error bars represent SD in technical replicates. For PR_87381 and PR_78074, bars represent estimates of the relative expression levels according to microarray mRNA profiling

Fig. 3

Wt re-isolates from oak became differentiated from California bay isolates on race tubes. Growth rates of triplicates of California bay isolates a Pr-1556 and b Pr-1557 were relatively constant for 18 weeks. Growth rates of triplicates of two re-isolates derived from each of c Pr-1556 and d Pr-1557 were also relatively constant in the first eight weeks (growth rates of the bay isolates and re-isolates are not significantly different between the 2nd and 9th weeks, one-way ANOVA p = 1). However, fluctuation in growth rates became apparent after ten weeks in the race tube (between 10 and 18th weeks, growth rates of bay isolates and re-isolates were significantly different, one-way ANOVA p = 3.5 × 10⁻³)

Fig. 4

Read-depth analyses revealed chromosomal aberrations in nwt isolates. Large CCNVs were revealed by BIC-seq analysis (upper graph for each panel) and by a read-depth analysis for heterozygous allele ratios using 10 KB long non-overlapping sliding window (lower graph). A concatenated view of the 52 largest scaffolds with a total length of 300 MB, corresponding to approximately half of the total genome of P. ramorum is shown. Scaffold numbers for large CCNV regions are indicated with pink bars, and those for LOH are shown with green bars. Scales show log (base 2) fold difference between sample isolates and reference isolates for BIC-seq analysis and log (base 2) ratios of alleles of sample isolates for the heterozygous allele frequency analysis. At each heterozygous locus, a read count ratio (more-abundant allele/less-abundant allele) was calculated. Allele count ratios larger than eight were set to eight to visualize the loss of heterozygosity. For the re-isolates Pr745#3 and Pr1156#7#1, corresponding progenitor isolates were used as reference for BIC-seq analyses (reference isolates listed in Table 3). a Pr-1556, an example of CCNV profile for the category “normal euploid”. b Nwt re-isolate Pr-745#3 from oak, an example of 3x CCNV, showing trisomy (1.5-fold increase in copy number) in the seven scaffolds. c Wt oak isolate Pr-140.9 (1x CCNV type) showing monosomy (0.5-fold decrease in copy number) in three scaffolds. d Nwt oak isolate MK516a, an example of copy number neutral LOH (2x cnLOH). Close inspection of short segments with CCNV seen as spikes in cnLOH regions (above the green bars) reveal the wt reference genome used for the BIC-seq analysis has heterozygous indels (>100 bp) in these regions. Loss of chromosomal segments harboring these indels in the nwt isolate MK516a resulted in spikes in the BIC-seq analysis. e A re-isolate from the race tube showed a mixture of nuclei with heterogeneous CCNVs. f A nwt EU1 isolate P2346 revealed extensive CCNVs and LOH when wt EU1 isolate P2363 was used as a reference

Fig. 5

Chromosomal breakpoints were associated with transposable elements. Of the nine NA1 isolates carrying chromosomal aberrations, a total of seven independent chromosomal breakpoints were identified at four chromosomal locations (see Additional files 4, 5, 6 and 7 for details). Black vertical lines represent transposable elements visualized by Integrative Genomics Viewer [81]. Cyan and orange bars represent segments of homologous chromosomes. a In scaffold 12, chromosomal breakpoints resulted in a partial monosomy in Pr-140.9 and a cnLOH in Pr-16. Two LINE retrotransposons were located at the breakpoint (red asterisk). b In scaffold 34, the chromosomal breakpoints for the partial trisomy (Pr-102 and Pr-16) and cnLOH (MK516a) occurred at the same genomic location. Four gypsy retrotransposons were found at the breakpoint (red asterisk). c Another breakpoint for cnLOH in Pr-16 was found in scaffold 37. Two gypsy retrotransposons flank the breakpoint (red asterisks). d The third breakpoint for cnLOH in Pr-16 was located in scaffold 44. A MuDR DNA transposon was located at the breakpoint (red asterisk). Additional files. 4, 5, 6 and 7 show close up of breakpoints