Characterization of resistance to pine wood nematode infection in Pinus thunbergiiusing suppression subtractive hybridization

Tomonori Hirao,Eitaro Fukatsu,Atsushi Watanabe

doi:10.1186/1471-2229-12-13

Tomonori Hirao, Eitaro Fukatsu + Show 1 more

Open Access

https://doi.org/10.1186/1471-2229-12-13

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Abstract

BackgroundPine wilt disease is caused by the pine wood nematode, Bursaphelenchus xylophilus, which threatens pine forests and forest ecosystems worldwide and causes serious economic losses. In the 40 years since the pathogen was identified, the physiological changes occurring as the disease progresses have been characterized using anatomical and biochemical methods, and resistant trees have been selected via breeding programs. However, no studies have assessed the molecular genetics, e.g. transcriptional changes, associated with infection-induced physiological changes in resistant or susceptible trees.ResultsWe constructed seven subtractive suppression hybridization (SSH) cDNA libraries using time-course sampling of trees inoculated with pine wood nematode at 1, 3, or 7 days post-inoculation (dpi) in susceptible trees and at 1, 3, 7, or 14 dpi in resistant trees. A total of 3,299 sequences was obtained from these cDNA libraries, including from 138 to 315 non-redundant sequences in susceptible SSH libraries and from 351 to 435 in resistant SSH libraries. Using Gene Ontology hierarchy, those non-redundant sequences were classified into 15 subcategories of the biological process Gene Ontology category and 17 subcategories of the molecular function category. The transcriptional components revealed by the Gene Ontology classification clearly differed between resistant and susceptible libraries. Some transcripts were discriminative: expression of antimicrobial peptide and putative pathogenesis-related genes (e.g., PR-1b, 2, 3, 4, 5, 6) was much higher in susceptible trees than in resistant trees at every time point, whereas expression of PR-9, PR-10, and cell wall-related genes (e.g., for hydroxyproline-rich glycoprotein precursor and extensin) was higher in resistant trees than in susceptible trees at 7 and 14 dpi.ConclusionsFollowing inoculation with pine wood nematode, there were marked differences between resistant and susceptible trees in transcript diversity and the timing and level of transcripts expressed in common; in particular, expression of stress response and defense genes differed. This study provided new insight into the differences in the physiological changes between resistant and susceptible trees that have been observed in anatomical and biochemical studies.

Highlights

Pine wilt disease is caused by the pine wood nematode, Bursaphelenchus xylophilus, which threatens pine forests and forest ecosystems worldwide and causes serious economic losses
The overlap between the two libraries at each time point was extremely low; for example, 0.4% of the cDNA inserts were shared between libraries from resistant and susceptible trees sampled at 1 dpi
Only 3.7% of the cDNA inserts were shared by the library from resistant trees sampled at 14 dpi and from susceptible trees sampled at 7 dpi

Summary

Introduction

Pine wilt disease is caused by the pine wood nematode, Bursaphelenchus xylophilus, which threatens pine forests and forest ecosystems worldwide and causes serious economic losses. Pine wilt disease is a chronic problem in pine forests In this breeding project, trees are screened for resistance using an artificial inoculation test that follows a strict protocol; during the first breeding program, which ran from 1978 to 1984, 92 resistant P. densiflora individuals were selected from 11,000 candidate trees, and only 16 resistant P. thunbergii individuals were selected from 14,000 candidate trees. Average rates of survival of openly pollinated progeny from the selected pines (i.e., resistant trees) were 64% for P. densiflora and 53% for P. thunbergii, which was respectively 16% and 40% higher than for unselected populations [4]

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