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Transcriptome analysis of the white pine blister rust pathogen Cronartium ribicola: de novo assembly, expression profiling, and identification of candidate effectors.

Liu JJ, Sturrock RN, Sniezko RA, Williams H, Benton R, Zamany A - BMC Genomics (2015)

Bottom Line: This study further identified a repertoire of candidate effectors and other pathogenicity determinants.This comprehensive transcriptome profiling substantially improves our current understanding of molecular WP-BR interactions.The repertoire of candidate effectors and other putative pathogenicity determinants identified here are valuable for future functional analysis of Cri virulence and pathogenicity.

View Article: PubMed Central - PubMed

Affiliation: Pacific Forestry Centre, Canadian Forest Service, Natural Resources Canada, 506 West Burnside Road, Victoria, BC, V8Z 1M5, Canada. Jun-Jun.Liu@NRCan-RNCan.gc.ca.

ABSTRACT

Background: The fungus Cronartium ribicola (Cri) is an economically and ecologically important forest pathogen that causes white pine blister rust (WPBR) disease on five-needle pines. To cause stem cankers and kill white pine trees the fungus elaborates a life cycle with five stages of spore development on five-needle pines and the alternate host Ribes plants. To increase our understanding of molecular WP-BR interactions, here we report genome-wide transcriptional profile analysis of C. ribicola using RNA-seq.

Results: cDNA libraries were constructed from aeciospore, urediniospore, and western white pine (Pinus monticola) tissues post Cri infection. Over 200 million RNA-seq 100-bp paired-end (PE) reads from rust fungal spores were de novo assembled and a reference transcriptome was generated with 17,880 transcripts that were expressed from 13,629 unigenes. A total of 734 unique proteins were predicted as a part of the Cri secretome from complete open reading frames (ORFs), and 41 % of them were Cronartium-specific. This study further identified a repertoire of candidate effectors and other pathogenicity determinants. Differentially expressed genes (DEGs) were identified to gain an understanding of molecular events important during the WPBR fungus life cycle by comparing Cri transcriptomes at different infection stages. Large-scale changes of in planta gene expression profiles were observed, revealing that multiple fungal biosynthetic pathways were enhanced during mycelium growth inside infected pine stem tissues. Conversely, many fungal genes that were up-regulated at the urediniospore stage appeared to be signalling components and transporters. The secreted fungal protein genes that were up-regulated in pine needle tissues during early infection were primarily associated with cell wall modifications, possibly to mask the rust pathogen from plant defenses.

Conclusion: This comprehensive transcriptome profiling substantially improves our current understanding of molecular WP-BR interactions. The repertoire of candidate effectors and other putative pathogenicity determinants identified here are valuable for future functional analysis of Cri virulence and pathogenicity.

No MeSH data available.


Related in: MedlinePlus

Venn diagram depicting transcripts expressed in Cronartium ribicola samples using RNA-seq. Numbers of contigs are indicated for the samples at the aeciospore stage collected from stem blisters of western white pine, at the urediniospore stage collected from infected Ribes leaves, and during in-planta mycelium growth from infected western white pine stems about 14 months after inoculation
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Fig3: Venn diagram depicting transcripts expressed in Cronartium ribicola samples using RNA-seq. Numbers of contigs are indicated for the samples at the aeciospore stage collected from stem blisters of western white pine, at the urediniospore stage collected from infected Ribes leaves, and during in-planta mycelium growth from infected western white pine stems about 14 months after inoculation

Mentions: To investigate in planta rust fungal gene expression, trimmed RNA-seq reads were mapped to the Cri reference transcriptome. 86 % of the reads from aeciospores and 85 % of the reads from urediniospores were aligned to the transcripts while about 24 % and 6 % of the reads from infected stem and infected needle tissues respectively were derived from the Cri reference transcriptome (Table 1). Of the Cri reference transcriptome, 13,591 (76 %), 14,022 (78 %) and 13,799 (77 %) transcripts were detected at life cycle stages of aeciospore, urediniospore, and infected stem, respectively. In total, we detected 10,972 transcripts that were commonly expressed in all three types of samples. In contrast, only 864, 960, and 800 contigs were detected with exclusive expression in aeciospore, urediniospore, and infected stem respectively (Fig. 3). To further analyze the detailed infection regulatory program, RNA-seq analysis was performed for four pair-wise comparisons of transcriptome profiles: aeciospore vs. urediniospore, aeciospore vs. infected stem, urediniospore vs. infected stem, and susceptible needles vs. resistant needles infected by basidiospores at 4 days post infection (4 dpi). The full set of expression data (FPKM) for the Cri reference transcriptome is shown in Additional file 1: Table S8. Pearson correlation analysis showed good sample reproducibility for aeciospores (R = 0.98 ± 0.02), urediniospores (R = 0.94 ± 0.04), and infected stems (R = 0.87 ± 0.10), respectively. Transcripts with normalized fold change > ±2 (p <0.05 after adjustment with false discovery rate - FDR) in at least one paired comparison were considered differentially expressed genes (DEGs) with up- or down-regulation and their numbers are shown for each comparison in Fig. 4. As compared to both aeciospore and urediniospore, in planta mycelium growth inside infected stem tissues consistently triggered a higher number of up-regulated transcripts than down-regulated transcripts (936 vs. 705).Table 1


Transcriptome analysis of the white pine blister rust pathogen Cronartium ribicola: de novo assembly, expression profiling, and identification of candidate effectors.

Liu JJ, Sturrock RN, Sniezko RA, Williams H, Benton R, Zamany A - BMC Genomics (2015)

Venn diagram depicting transcripts expressed in Cronartium ribicola samples using RNA-seq. Numbers of contigs are indicated for the samples at the aeciospore stage collected from stem blisters of western white pine, at the urediniospore stage collected from infected Ribes leaves, and during in-planta mycelium growth from infected western white pine stems about 14 months after inoculation
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4559923&req=5

Fig3: Venn diagram depicting transcripts expressed in Cronartium ribicola samples using RNA-seq. Numbers of contigs are indicated for the samples at the aeciospore stage collected from stem blisters of western white pine, at the urediniospore stage collected from infected Ribes leaves, and during in-planta mycelium growth from infected western white pine stems about 14 months after inoculation
Mentions: To investigate in planta rust fungal gene expression, trimmed RNA-seq reads were mapped to the Cri reference transcriptome. 86 % of the reads from aeciospores and 85 % of the reads from urediniospores were aligned to the transcripts while about 24 % and 6 % of the reads from infected stem and infected needle tissues respectively were derived from the Cri reference transcriptome (Table 1). Of the Cri reference transcriptome, 13,591 (76 %), 14,022 (78 %) and 13,799 (77 %) transcripts were detected at life cycle stages of aeciospore, urediniospore, and infected stem, respectively. In total, we detected 10,972 transcripts that were commonly expressed in all three types of samples. In contrast, only 864, 960, and 800 contigs were detected with exclusive expression in aeciospore, urediniospore, and infected stem respectively (Fig. 3). To further analyze the detailed infection regulatory program, RNA-seq analysis was performed for four pair-wise comparisons of transcriptome profiles: aeciospore vs. urediniospore, aeciospore vs. infected stem, urediniospore vs. infected stem, and susceptible needles vs. resistant needles infected by basidiospores at 4 days post infection (4 dpi). The full set of expression data (FPKM) for the Cri reference transcriptome is shown in Additional file 1: Table S8. Pearson correlation analysis showed good sample reproducibility for aeciospores (R = 0.98 ± 0.02), urediniospores (R = 0.94 ± 0.04), and infected stems (R = 0.87 ± 0.10), respectively. Transcripts with normalized fold change > ±2 (p <0.05 after adjustment with false discovery rate - FDR) in at least one paired comparison were considered differentially expressed genes (DEGs) with up- or down-regulation and their numbers are shown for each comparison in Fig. 4. As compared to both aeciospore and urediniospore, in planta mycelium growth inside infected stem tissues consistently triggered a higher number of up-regulated transcripts than down-regulated transcripts (936 vs. 705).Table 1

Bottom Line: This study further identified a repertoire of candidate effectors and other pathogenicity determinants.This comprehensive transcriptome profiling substantially improves our current understanding of molecular WP-BR interactions.The repertoire of candidate effectors and other putative pathogenicity determinants identified here are valuable for future functional analysis of Cri virulence and pathogenicity.

View Article: PubMed Central - PubMed

Affiliation: Pacific Forestry Centre, Canadian Forest Service, Natural Resources Canada, 506 West Burnside Road, Victoria, BC, V8Z 1M5, Canada. Jun-Jun.Liu@NRCan-RNCan.gc.ca.

ABSTRACT

Background: The fungus Cronartium ribicola (Cri) is an economically and ecologically important forest pathogen that causes white pine blister rust (WPBR) disease on five-needle pines. To cause stem cankers and kill white pine trees the fungus elaborates a life cycle with five stages of spore development on five-needle pines and the alternate host Ribes plants. To increase our understanding of molecular WP-BR interactions, here we report genome-wide transcriptional profile analysis of C. ribicola using RNA-seq.

Results: cDNA libraries were constructed from aeciospore, urediniospore, and western white pine (Pinus monticola) tissues post Cri infection. Over 200 million RNA-seq 100-bp paired-end (PE) reads from rust fungal spores were de novo assembled and a reference transcriptome was generated with 17,880 transcripts that were expressed from 13,629 unigenes. A total of 734 unique proteins were predicted as a part of the Cri secretome from complete open reading frames (ORFs), and 41 % of them were Cronartium-specific. This study further identified a repertoire of candidate effectors and other pathogenicity determinants. Differentially expressed genes (DEGs) were identified to gain an understanding of molecular events important during the WPBR fungus life cycle by comparing Cri transcriptomes at different infection stages. Large-scale changes of in planta gene expression profiles were observed, revealing that multiple fungal biosynthetic pathways were enhanced during mycelium growth inside infected pine stem tissues. Conversely, many fungal genes that were up-regulated at the urediniospore stage appeared to be signalling components and transporters. The secreted fungal protein genes that were up-regulated in pine needle tissues during early infection were primarily associated with cell wall modifications, possibly to mask the rust pathogen from plant defenses.

Conclusion: This comprehensive transcriptome profiling substantially improves our current understanding of molecular WP-BR interactions. The repertoire of candidate effectors and other putative pathogenicity determinants identified here are valuable for future functional analysis of Cri virulence and pathogenicity.

No MeSH data available.


Related in: MedlinePlus