On the species of origin: diagnosing the source of symbiotic transcripts

Abstract

Background: Most organisms have developed ways to recognize and interact with other species. Symbiotic interactions range from pathogenic to mutualistic. Some molecular mechanisms of interspecific interaction are well understood, but many remain to be discovered. Expressed sequence tags (ESTs) from cultures of interacting symbionts can help identify transcripts that regulate symbiosis, but present a unique challenge for functional analysis. Given a sequence expressed in an interaction between two symbionts, the challenge is to determine from which organism the transcript originated. For high-throughput sequencing from interaction cultures, a reliable computational approach is needed. Previous investigations into GC nucleotide content and comparative similarity searching provide provisional solutions, but a comparative lexical analysis, which uses a likelihood-ratio test of hexamer counts, is more powerful.

Results: Validation with genes whose origin and function are known yielded 94% accuracy. Microbial (non-plant) transcripts comprised 75% of a Phytophthora sojae-infected soybean (Glycine max cv Harasoy) library, contrasted with 15% or less in root tissue libraries of Medicago truncatula from axenic, Phytophthora medicaginis-infected, mycorrhizal, and rhizobacterial treatments. Mycorrhizal libraries contained about 23% microbial transcripts; an axenic plant library contained a similar proportion of putative microbial transcripts.

Conclusions: Comparative lexical analysis offers numerous advantages over alternative approaches. Many of the transcripts isolated from mixed cultures were of unknown function, suggesting specificity to symbiotic metabolism and therefore candidates likely to be interesting for further functional investigation. Future investigations will determine whether the abundance of non-plant transcripts in a pure plant library indicates procedural artifacts, horizontally transferred genes, or other phenomena.

Figure 1

Distribution of GC content in pure and mixed-culture libraries. (a) Probability densities for histogram bin sizes of 0.02 (2%) in base content. (b) Cumulative probability distribution functions (cdfs).

Figure 2

Distribution of hexamer dissimilarity test results from pure and mixed-culture libraries. (a) Calculation of statistical parameters from cdfs A and B. Overlap in the upper tail of cdf_A with cdf_B and the lower tail of cdf_B with cdf_A are likely regions for error. We find the false-positive rate α where 1 - cdf_A intersects 0 [cdf_A(0)= 1 - α], and the false-negative rate β where cdf_B crosses 0. Also shown are the medians (μ) for each distribution, where cdf(μ) = 0.5. (b) Calibration curves for plant (A₁, Glycine and Medicago spp., solid black line) and stramenopile plus P. infestans EST (B₁, dashed black line) training sequences. Superimposed distributions of test results show dissimilarity differences for infected G. max (green) and axenic P. sojae mycelial and zoospore sequences (blue and cyan, respectively).

Figure 3

Paired dissimilarity test results from pure and mixed-culture libraries. Each point corresponds to an expressed tag from either (a) infected G. max or (b) axenic P. sojae mycelial or (c) zoospore sequences, compared with plant (A₁) and stramenopile plus P. infestans EST training sequences (B₁). The identity function indicates equal dissimilarity to both training sets, t=D(A) - D(B)= 0. Points above the identity function are more plant-like than points below.

Figure 4

Dissimilarity distributions from Medicago truncatula libraries. Calibration curves compare plant training sets (A₁ and A₂, solid black lines) with one of three microbial symbiont training sets (broken black lines): (a) Stramenopile and P. infestans EST sequences (B₁); (b) pooled zygomycete and chytridiomycete coding sequences (B₂); and (c) sequences from the genera Rhizobium, Sinorhizobium and Bradyrhizobium (B₃). Cumulative distributions of test results from M. truncatula axenic and microbial symbiont mixed cultures appear in each panel (colored lines).

Figure 5

Paired comparison results frompure and mixed-culture M. truncatula libraries. Each point indicates the dissimilarity of a test sequence compared with a plant training set (A₁ or A₂) and one of three microbial symbiont training sets: (a) Stramenopile and P. infestans EST sequences (B₁); (b) pooled zygomycete and chytridiomycete coding sequences (B₂); and (c) sequences from the genera Rhizobium, Sinorhizobium and Bradyrhizobium (B₃). Sequences from M. truncatula axenic (green) and microbial symbiont mixed culture libraries are represented in each panel. The identity function (y = x) is also shown.