Abstract
Pine wilt disease, caused by the pinewood nematode, Bursaphelenchus xylophilus, is one of the world’s most serious tree diseases. Although the B. xylophilus whole-genome sequence and comprehensive secretome profile have been determined over the past decade, it remains unclear what molecules are critical in pine wilt disease and govern B. xylophilus virulence in host pine trees. Here, a comparative secretome analysis among four isolates of B. xylophilus with distinct virulence levels was performed to identify virulence determinants. The four candidate virulence determinants of B. xylophilus highly secreted in virulent isolates included lipase (Bx-lip1), glycoside hydrolase family 30 (Bx-GH30), and two C1A family cysteine peptidases (Bx-CAT1 and Bx-CAT2). To validate the quantitative differences in the four potential virulence determinants among virulence groups at the protein level, we used real-time reverse-transcription polymerase chain reaction analysis to investigate these determinants at the transcript level at three time points: pre-inoculation, 3 days after inoculation (dai), and 7 dai into pine seedlings. The transcript levels of Bx-CAT1, Bx-CAT2, and Bx-GH30 were significantly higher in virulent isolates than in avirulent isolates at pre-inoculation and 3 dai. A subsequent leaf-disk assay based on transient overexpression in Nicotiana benthamiana revealed that the GH30 candidate virulent factor caused cell death in the plant. Furthermore, we demonstrated that Bx-CAT2 was involved in nutrient uptake for fungal feeding via soaking-mediated RNA interference. These findings indicate that the secreted proteins Bx-GH30 and Bx-CAT2 contribute to B. xylophilus virulence in host pine trees and may be involved in pine wilt disease.
Highlights
Pine wilt disease, caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus, is one of the most serious tree diseases worldwide because pine forests are essential sources of forest products
We identified four proteins that were highly expressed in two virulent isolates: class 3 lipase (Bx-lip1), glycosyl hydrolase family 30 (Bx-glycoside hydrolase family 30 (GH30)), cathepsin L (Bx-CAT1), and cathepsin L (Bx-CAT2) (Table 1)
These label-based methods are generally considered more sensitive and comprehensive compared to the gel-based methods used in our study, we presume that we have identified the major differentially secreted proteins between virulent and avirulent isolates
Summary
Pine wilt disease, caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus, is one of the most serious tree diseases worldwide because pine forests are essential sources of forest products. Pine forests face a catastrophic threat from pine wilt disease in East Asian countries. In the 1980s, the pathogen spread to neighboring East Asian countries (Yi et al, 1989), and was subsequently isolated in Portugal in 1999 (Mota et al, 1999) and in Spain in 2008 (Abelleira et al, 2011). Because of active international trade in forest products, the PWN poses a potentially serious problem in both the Southern and Northern Hemispheres. The PWN is currently a worldwide threat
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