Abrantes, I.

Duarte, T.

Egas, C.

Fonseca, L.

Gomes, P.

Leisico, F.

Pinheiro, M.

English

JKI Open Journal Systems (Julius Kühn-Institut)

  IUFRO / REPHRAME International Conference on Pine Wilt Disease 2013  Berichte aus dem Julius Kühn-Institut 169. 2013 87 Egas et al. Comparative transcriptomics to understand the molecular basis of Bursaphelenchusxylophilus pathogenicity. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 87-89, Braunschweig, ISSN: 1866-590X Comparative transcriptomics to understand the molecular basis of Bursaphelenchusxylophilus pathogenicity Egas C11 Genoinseq, Biocant Park, 3060-197 Cantanhede, Portugal;  2 Department of Chemistry University of Aveiro, Campus de Santiago, 3810-193 Aveiro, PT 3 Department of Life Sciences, University of Coimbra, 3001-401 Coimbra, Portugal , Leisico F2, Duarte T1, Gomes P1, Fonseca L3, Abrantes I3, Pinheiro M1. Email: cegas@biocant.pt  Pinewood nematode (PWN) biology and ecology are strictly associated to pine wilt disease (PWD) and have been extensively investigated. However, the disease molecular mechanism has not yet been established. Aiming to unravel the mechanism of pathogenicity we used a transcriptomics approach to study the gene expression of Bursaphelenchus xylophilus and the closely related Bursaphelenchus mucronatus. Furthermore, we sequenced the transcriptomes of B. xylophilus males, females and dispersal juveniles JIII. We then built a transcriptomics platform to carry out educated searches on differential gene expression to highlight the molecular basis of PWN pathogenicity or discover new targets with high interest for nematode control.  The five transcriptomes were sequenced in the 454 platform (Roche). Pyrosequencing generated on average 455,000 reads and 8,500 transcripts per transcriptome; more than 60% of these corresponded to InterPro terms (Table 1). Nucleotide and aminoacid sequences and corresponding annotation were organized in a web-based database. The huge amount of data generated represents an important opportunity to increase the available scientific knowledge on the nematode and to carry out comparative analysis.  Table 1. Summary of sequencing, assembly and annotation data. B. xylophilus and B. mucronatus mixed stages were collected from fungal cultures, while B. xylophilus males, females and JIII were collected from infected pines. Total RNA was isolated from each nematode isolate, and cDNA synthesized according to the SMART technology. Transcript assembly and annotation were performed as described in Bettencourt (2010).   B. xylophilus (fungi) B. mucronatus (fungi) B. xylophilus (pine)  male female JIII # Reads 647,641 465,256 407,835 227,307 531,049 # Transcripts 11,006 8,822 6,724 5,760 10,608 # Amino acid sequences 12,038 9,231 6,897 6,013 11,444 # Amino acid sequences   assigned to InterPro  7,321 5,547 4,148 4,135 7,120   IUFRO / REPHRAME International Conference on Pine Wilt Disease 2013  Berichte aus dem Julius Kühn-Institut 169. 2013 88  The platform was queried for three main comparisons: B. xylophilus versus B. mucronatus grown in fungi; PWN males versus females versus JIII grown on pine and PWN grown on fungi versus growth in pine. The transcriptomes were compared for genes exclusively present in each condition according to different annotation strategies such as KEGG and Gene Ontology, and also based on sequences similarity using the CD-HIT program (Huang 2010). In addition, we also studied the gene expression of potential nematode parasitism effetors directly on the database using Myrna as described in Santos (2012). The comparisons identified more than 30 genes potentially involved in PWD parasitism that are being experimentally validated by RT-PCR. Here, we focus on the comparative gene expression of oxidative response genes, ubiquitination-related genes and a potential secreted purple acid phosphatase (Figure 1). The gene expression experimental validation indicated an overexpression in the oxidative response in males and JIII when compared to females grown in pine and also an overexpression in B. mucronatus when compared to B. xylophilus grown on fungi. Interestly, only B. xylophilus males showed an overexpression of 4HPPD (4-hydroxyphenylpyruvate dioxygenase) and an underexpression of HGD (homogentisic acid oxidase), indicating that also homogentisate can be a potentially important nematode oxidative detoxification mechanism in pine (Martin and Batkoff, 1987; Arias-Barrau, 2004). Ubiquitination-related genes were overexpressed in females when compared to the other B. xylophilus samples, suggesting an important regulation of internal proteolytic activity, probably to reach normal homeostasis required for reproduction (Comyn, 2013). The marked overexpression of secreted PAP (purple acid phosphatase) in females and JIII when compared to B. xylophilus grown in fungi and males grown in pine may represent a potential role in the nematode interaction with pine due to the relation of PAPs to a wide range of actions such as reactive oxygen species generation (Schenk, 2013).   Figure 1. Differential gene expression determined by RT-PCR. Gluthathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT) were studied in the five conditions, while 4-hydroxyphenylpyruvate dioxygenase (4HPPD), homogentisic acid oxidase (HGD), Ubiquitin-specific-processing protease 33 (Usp 33), Ubiquitin-specific-processing protease 14 (Usp 14) and purple acid phosphatase (PAP) were studied in the four B. xylophilus samples. Data is presented under the form mean±SD (standard deviation). * indicates significative differential expression with p < 0.05 as determined with the REST software. The cell division control protein 42 was the endogenous control and B. xylophilus grown in fungi the control sample.     IUFRO / REPHRAME International Conference on Pine Wilt Disease 2013  Berichte aus dem Julius Kühn-Institut 169. 2013 89 Additional differentialy expressed genes corresponded to peptidases and respective inhibitors, carbohydrate-active enzymes, genes involved in oxidative detoxification, phenolic compound degradation or host mimicking. These genes showed higher expression levels for the nematode grown in pine, and also differences between the 3 developmental stages. These results suggest differences between males, females and JIII while growing in pine and may elucidate the contribution of the different stages to the PWN pathogenicity. Results and discussion of these genes will be presented at the meeting. REFERENCES  Arias-Barrau E; Olivera ER; Luengo JM; Fernández C; Galán B; García JL; Díaz E; Miñambres B (2004). The homogentisate pathway: a central catabolic pathway involved in the degradation of L-phenylalanine, L-tyrosine, and 3-hydroxyphenylacetate in Pseudomonas putida. Journal of Bacteriology 186, 5062-5077. Bettencourt R; Pinheiro M; Egas C; Gomes P; Afonso M; Shank T; Santo RS (2010). High-throughput sequencing and analysis of the gill tissue transcriptome from the deep-sea hydrothermal vent mussel Bathymodiolus azoricus. BMC genomics 11, 559. Comyn S; Chan G; Mayor T (2013). False start: Cotranslational protein ubiquitination and cytosolic protein quality control. Journal of Proteomics (0). Huang Y; Niu B; Gao Y; Li W (2010). CD-HIT Suite: a web server for clustering and comparing biological sequences. Bioinformatics 26(5), 680-682.  Martin Jr JP; Batkoff B (1987). Homogentisic acid autoxidation and oxygen radical generation: implications for the etiology of alkaptonuric arthritis. Free Radic Biol Med 3, 241–250. Santos CS; Pinheiro M; Silva AI; Egas C; Vasconcelos MW (2012). Searching for resistance genes to Bursaphelenchus xylophilus using high throughput screening. BMC Genomics 13, 599. Schenk G; Mitić N; Hanson GR; Comba P (2013). Purple acid phosphatase: A journey into the function and mechanism of a colorful enzyme. Coordination Chemistry Reviews 257(2), 473-482.   

Comparative transcriptomics to understand the molecular basis of Bursaphelenchus xylophilus pathogenicity

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Comparative transcriptomics to understand the molecular basis of Bursaphelenchus xylophilus pathogenicity

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