Proteomic analysis of Masson pine with high resistance to pine wood nematodes

Jingbin GAO; Ting PAN; Xuelian CHEN; Qiang Wei; Liuyi Xu

doi:10.1371/journal.pone.0273010

. 2022 Aug 12;17(8):e0273010. doi: 10.1371/journal.pone.0273010

Proteomic analysis of Masson pine with high resistance to pine wood nematodes

Jingbin GAO ^1,², Ting PAN ^2,³, Xuelian CHEN ^2,³, Qiang Wei ^2,³, Liuyi Xu ^2,^3,^*

Editor: Jon M Jacobs⁴

PMCID: PMC9374249 PMID: 35960732

Abstract

Pine wilt disease is a dangerous pine disease globally. We used Masson pine (Pinus massoniana) clones, selected through traditional breeding and testing for 20 years, to study the molecular mechanism of their high resistance to pine wood nematodes (PWN,Bursaphelenchus xylophilus). Nine strains of seedlings of genetically stable Masson pine screened from different families with high resistance to PWN were used. The same number of sensitive clones were used as susceptible controls. Total proteins were extracted for tandem mass tag (TMT) quantitative proteomic analysis. The key proteins were verified by parallel reaction monitoring (PRM). A threshold of upregulation greater than 1.3-fold or downregulation greater than 0.3-fold was considered significant in highly resistant strains versus sensitive strains. A total of 3491 proteins were identified from the seedling tissues, among which 2783 proteins contained quantitative information. A total of 42 proteins were upregulated and 96 proteins were downregulated in the resistant strains. Functional enrichment analysis found significant differences in the proteins with pectin esterase activity or peroxidase activity. The proteins participating in salicylic acid metabolism, antioxidant stress reaction, polysaccharide degradation, glucose acid ester sheath lipid biosynthesis, and the sugar glycosaminoglycan degradation pathway were also changed significantly. The PRM results showed that pectin acetyl esterase, carbonic anhydrase, peroxidase, and chitinase were significantly downregulated, while aspartic protease was significantly upregulated, which was consistent with the proteomic data. These results suggest that Masson pine can degrade nematode-related proteins by increasing protease to inhibit their infestation, and can enhance the resistance of Masson pine to PWN by downregulating carbon metabolism to limit the carbon available to PWN or for involvement in cell wall components or tissue softening. Most of the downregulated proteins are supposed to act as an alternative mechanism for latter enhancement after pathogen attacks. The highly resistant Masson pine, very likely, harbors multiple pathways, both passive and active, to defend against PWN infestation.

Introduction

Pine wilt disease (PWD) is a dangerous pine disease worldwide [1], occurring in many countries. Since it spread to China, it has infested many species of Pinus. Initially, it affected black pine (Pinus thunbergii), and it now mainly affects Masson pine (P. massoniana), which is a pioneer tree species for the greening of barren hills. Masson pine plays an important role in maintaining the safety of the ecological environment and natural landscape, and in soil and water conservation [2].

At present, PWD is mainly prevented by the application of quarantine and chemical pesticides to control pine wood nematodes (PWNs; Bursaphelenchus xylophilus) and to kill Monochamus alternatus that is an insect vector. Although these methods can slow the rapid spread of PWD, chemical prevention and control measures cause pollution to the environment, making it very difficult to apply pesticides on a large scale, and the cost of prevention is very high. Tree felling and trapping of M. alternatus have also worked well. A slow initial response to the discovery of PWN hindered attempts to limit its outward spread; decisive action is needed in the crucial early stages of an outbreak. Recently, fluopyram was found to have significant nematocidal activity against B. xylophilus as a new trunk-injection agent [3]. It is very difficult to avoid the occurrence and emergence of new epidemic areas year after year. Because the pathogenesis of PWD and the complexity of its propagation media are not yet fully understood, there are no particularly effective control measures yet. Therefore, the question of how to effectively prevent and control PWD has become urgent in forestry production and management [2]. The sustainable development policies for forests include new regulations for environmentally friendly and cost-effective PWD prevention and control. The selective breeding of PWN-resistant Masson pine is an optimal choice and has attracted more and more attention [4]. Research on the resistance breeding of black pine (P. thunbergii) and red pine (P. densiflora) in Japan has shown that the breeding of resistant varieties is a very effective measure in the comprehensive prevention and control of PWD [5].

The occurrence and development of PWD are the result of a combination of multiple biological factors such as pine trees, vector insects, PWN, and related microorganisms (https://www.bspp.org.uk/wp-content/uploads/2021/01/PWD-final-report-1.1EB.pdf). Scientists have tried to find ways to prevent and control PWD based on the physiology and biochemistry of pine trees [6, 7] and on characteristic studies of PWN [8]. The endophytic and rhizospheric microbial community changes are potentially caused by B. xylophilus infection in pines [9]. In contrast, the resident bacterial communities, such as Stenotrophomonas and Bacillus spp., exhibit significant inhibitory activities against PWN during their developmental stages [10], suggesting their potential as biocontrol agents to combat PWD. More precisely, Bacillus thuringiensis JCK-1233 has been found to control PWD by elicitation of a moderate hypersensitive reaction before infection to afford pines greater resistance [11].

Research on resistance mechanisms has been carried out from the perspective of resistance genes, with the goal of finding genes related to resistance [12–14]. Recently, maritime pine (P. pinaster) and Yunnan pine (P. yunnanensis) were compared in terms of resistance to PWN. The results showed that the response was less complex and involved a smaller number of differentially expressed genes in Yunnan pine, which may be associated with its increased degree of resistance to PWN [15]. In addition to the response to PWN infection [16], the species-specific physiology before the infection is very important in a plant’s defense strategy.

In this study, we used seedlings of Masson pine with high resistance to PWN and those from sensitive strains as controls. The resistant clones were obtained via classical screening and breeding methods. The proteomic data were collected and analyzed. The expression of many proteins involved in resistance changed and their involvement in resistance is discussed. Those results suggest that the highly resistant strains possess a network of multiple resistance pathways.

Materials and methods

Biological material, pine wood nematode inoculation, and sampling

In the PWD epidemic and nonepidemic areas in Anhui Province, China, in 2000, we collected the seeds of Masson pine trees from a total of 10 counties (Huangshan, Guangde, Jing, Xuanzhou, Xiuning, Nanqiao, He, Qianshan, Taihu, and Quanjiao) with the permission of the local government. In 2001, the seedlings were sown and cultivated in family groups in Hefei, Anhui (11720′E, 31°92′N; altitude, 35 m above sea level). Half of each family’s seedlings were transplanted into plastic greenhouses in the spring of 2003 [17]. PWN KS-3B, the most virulent isolate, was selected from 40 infected dead Masson pine in different locations. After 90 days of inoculation with PWN, the first evaluation was performed in July 2003. Seedlings were inoculated with a suspension in 0.5 mL of water containing a mixed population of 5000 nematodes. P. taeda and P. elliottii were set as the resistant control species. Healthy seedlings from 44,400 seedlings of 384 families were tested. Three to five seedlings were selected from each family as resistant candidates. A total of 1201 Masson pine resistant candidates from 251 families were selected, and these individuals were planted according to a row spacing of 2 × 3 m. In 2006, the shoots from the resistant candidates were grafted and P. elliottii was used as the rootstock. The second and third evaluation were performed for two consecutive years in July 2007 and July 2008 [18]. PWN was inoculated with a suspension in 0.5 mL of water containing a mixed population of 10,000 nematodes in the field and virulence was determined if the selected grafted seedlings were resistant each year.

According to the results of the clone resistance evaluation, the highly resistant single trees from different families were selected as the high-resistance strains, and the nonresistant single trees were used as the susceptible group. Nine strains were randomly selected from each group, and three of them were pooled as one biological sample, with a total of 3 biological replicates.

Tandem mass tag (TMT) quantitative proteome analysis

Sample preparation

Tender branches were taken from individual trees. The pine leaves, leaf sheaths, and periderm were removed from the branches, and the phloem was obtained and quickly frozen in liquid nitrogen for 1 min. The samples were kept on dry ice during transportation, and the samples were stored in a –80°C freezer for later use.

Protein extraction and trypsin digestion

The samples were first ground with liquid nitrogen, and then the powder was transferred to a 5 mL centrifuge tube and sonicated three times on ice using a high-intensity ultrasonic processor (Scientz) in lysis buffer (including 1% TritonX-100, 10 mM dithiothreitol, 1% protease inhibitor cocktail, 50 μM of PR-619, 3 μM of TSA, 50 mM NAM, 2 mM EDTA, and 1% phosphatase inhibitor for phosphorylation). An equal volume of Tris-saturated phenol (pH8.0) was added. The mixture was further vortexed for 5 min. After centrifugation (4°C, 10 min, 5000 g), the upper phenol phase was transferred to a new centrifuge tube. The proteins were precipitated by adding at least four volumes of ammonium-sulfate-saturated methanol and incubated at –20°C for at least 6 h. After centrifugation at 4°C for 10 min, the supernatant was discarded. The remaining precipitate was washed with ice-cold methanol, followed by washing three times with ice-cold acetone. The protein was redissolved in 8 M urea and the protein concentration was determined using a BCA kit according to the manufacturer’s instructions.

The sample was trypsinized according to the instructions from Jingjie PTM BioLab (Hangzhou) Co. Ltd. Finally, the peptides were desalted using a C18 SPE column.

TMT labeling

Tryptic peptides were first dissolved in 0.5 M TEAB. Each peptide channel was labeled with the respective TMT reagent (based on manufacturer’s protocol, Thermo Scientific) and incubated for 2 h at room temperature. Five microliters of each sample were pooled, desalted, and analyzed by MS to check the labeling efficiency. After the labeling efficiency check, the samples were quenched by adding 5% hydroxylamine. The pooled samples were then desalted with a Strata X C18 SPE column (Phenomenex) and dried by vacuum centrifugation.

HPLC fractionation

The sample was fractionated by high-pH reverse-phase HPLC using Agilent 300 Extend C18 column (5 μm particles, 4.6 mm ID, 250 mm length). Briefly, the peptides were separated with a gradient of 8%–32% acetonitrile in 10 mM ammonium bicarbonate (pH 9) over 64 min into 54 fractions. The peptides were then combined into nine fractions and dried by vacuum centrifugation.

LC–MS/MS analysis

Afterwards, the peptides were dissolved in the mobile phase A of liquid chromatography and then separated using the EASY-nLC 1200 ultra-high performance liquid system. The whole procedure was guided by Jingjie PTM BioLab (Hangzhou) Co. Ltd. The data acquisition mode uses the data-dependent scanning (DDA) program. In order to improve the effective utilization of the mass spectrometer, the automatic gain control (AGC) was set to 5E4, the signal threshold was set to 2.5 E5 ions/s, the maximum injection time was set to 40 ms, and the dynamic rejection time of the tandem mass spectrometry scanning was set to 30 s to avoid repeated scanning of precursor ions.

Database search and bioinformatics analysis

The database used was P. Massoniana (14,494 sequences), and a decoy database was added to calculate the false discovery rate (FDR) caused by random matching, while a common contamination database was added to the database to eliminate the influence of contaminated proteins in the identification results.

Protein annotation was carried out using Gene Ontology (GO) analysis, protein structural domain annotation, KEGG pathway annotation, and the subcellular location method, while cluster analysis based on the functional enrichment of proteins and protein interaction network-based analysis was carried out to determine the functional enrichment of proteins using GO enrichment analysis, pathway enrichment analysis, and protein structural domain enrichment analysis.

Quantitative verification of targeted proteome by mass spectrometry-based parallel reaction monitoring (PRM)

LC–MS analysis

After the peptides were separated by the ultra-high-performance liquid system, they were injected into the NSI ion source for ionization and then entered into the Q ExactiveTM Plus mass spectrometer for analysis. The ion source voltage was set to 2.1 kV, and both the peptide precursor ions and their secondary fragments were detected and analyzed using the high-resolution Orbitrap (Jingjie PTM BioLab (Hangzhou) Co. Ltd).

Proteomic statistical analysis of the data

First, the average quantitative value was calculated for the three repeats of each group, the resistant or susceptible. The value from the susceptible group was taken as a control. The ratio of the average values between the two groups was calculated and taken as the final differential expression levels. The log2 calculation was performed to gain the relative quantitative values, which conforms to a normal distribution. The two-tailed t-test method was used to calculate the p-value. A change of differential expression exceeding 1.3 was considered as a significant upregulation when the p-value <0.05, while a change less than 0.7 was considered as a significant downregulation when the p-value <0.05.

Results and discussion

Evaluation of the resistance of Masson pine clones to PWD

The seeds were collected in 2001 from Masson pine in endemic and nonendemic areas in Anhui provinces, respectively. The seedlings were first inoculated in 2003, and the susceptibility of the seedlings was evaluated 90 days after inoculation (Fig 1A). The second inoculation was carried out for the resistant strains in the same year. The susceptibility of the newly grafted seedlings was evaluated twice after inoculation in 2007 and 2008 (Fig 1B).

Fig 1 — (A) The phenotype 90 days post-inoculation of B. *xylophilus* in a plastic greenhouse in 2003. (B) The phenotype 90 days post field inoculation of B. *xylophilus* in 2007. The complete green indicates resistant individuals, while the withered dead plants were considered to be susceptible individuals.

General proteomic analysis

Quantitative proteomics mass spectrometric analysis was carried out for the Masson pine seedlings via TMT labeling, and a total of 342,006 secondary spectrograms were obtained. After the secondary spectrograms of the mass spectrometer were retrieved through the protein theoretical database, the number of available effective spectrograms was 33,220 and the utilization rate of the spectrograms was 9.7%. A total of 18,619 peptides were identified through spectrogram analysis, of which 17,715 were specific peptides. A total of 3491 proteins were identified, of which 2783 could be quantitatively analyzed. The statistical results showed that the biological repeatability between the samples was good enough, particularly for susceptible strains (Fig 2A). An overall distribution diagram of the differential proteins is presented as volcano plots. There were 138 differentially expressed proteins (R/S) obtained in total, of which 42 were upregulated (in red) and 96 were downregulated (in blue in Fig 2B). We then analyzed 17 redundant proteins from each sample for PRM verification and confirmed the data applicability for further analysis (one representative is shown in Fig 2C).

Fig 2 — (A) Two-dimensional scatter plots for analysis of the protein quantitative principal components among the replicate samples. Three statistical analysis methods, i.e., principal component analysis (PCA), relative standard deviation (RSD), and Pearson’s correlation coefficient, were used to evaluate the protein quantitative repeatability. S: susceptible seedlings; R: resistant seedlings. (B) Quantitative volcano plot of the differentially expressed proteins. The horizontal axis is the value of the relative quantitative value of the proteins after log2 logarithmic conversion, and the vertical axis is the value of the significance test p-value after -log10 logarithmic conversion. The red points indicate upregulation, while the blue points indicate downregulation. (C) A representative of parallel reaction monitoring (PRM) verification. A LAYPDIQIISNCDGSSK peak areas graph of the fragment ion was used as an example to confirm the data applicability for further analysis.

Functional classification of the differentially expressed proteins

The distribution of the differentially expressed proteins in the secondary annotations of GO is shown in Fig 3 and S1 and S2 Tables. The expression levels of proteins related to cell metabolism and respondence to external stimuli were very high, and the indexes classified by enzyme activity indicated upregulated and downregulated proteins with catalytic activities (Fig 3A and 3B).

Upon further analysis of the functional enrichment, enzymes such as oxidoreductase, pectinesterase inhibitor, pectinesterase, and carboxylate hydrolase, showed significant differential expression. Moreover, the proteins involved in these biological processes varied substantially not only in expression but also in the number of genes (Fig 4 and S3 Table). The similar results were obtained in agenomic transcriptional study, in which genes encoding pectate lyase were found upregulated in the resistant Masson pine compared with the susceptible clones [13]. It is speculated that changes in these genes might be involved in the resistance to PWD.

Fig 4 — The size of each circle stands for the number of differentially expressed proteins enriched in corresponding function. The rich factor (log2 fold enrichment) was calculated using the number of enriched genes divided by the number of all background genes in corresponding functions.

Analysis of the resistance network using the upregulated proteins

Disease resistance in plants depends on genes that are either expressed prior to infection (so-called passive) or induced to express post-infestation by pathogens (so-called active) so that they can defend themselves in a timely manner. Many resistance genes have been characterized in numerous plant species that collectively confer resistance to pathogens, including nematodes and insects [19], as an overall immune system [20]. As shown, we found 42 genes that were upregulated in resistant P. massoniana compared to vulnerable clones (S3 Table).

Of these, two aspartic proteinases (At4g16563), a pepsin-like aspartic protease, and aspartic proteinase 4, a saposin-like aspartyl protease family protein, were found to be upregulated by 90% and 39%, respectively, in resistantP. massoniana.The enzymes are mostly secreted from cells as inactive proenzymes that activate autocatalytically at an acidic pH. An apoplastic aspartic protease in Arabidopsis was identified to contribute to constitutive disease resistance, which is salicylic acid (SA)-dependent [21]. Though SA was not detected in resistant P. massoniana strains here, the highly expressed aspartic proteases suggest its big contribution to the defense system against PWN.

Plant nucleotide binding site–Leu-rich repeat (NBS-LRR) proteins are associated with the recognition of and resistance to pathogens, and represent the largest of five known classes of R proteins [22]. In our study, we found two LRR proteins, including plant intracellular Ras-group-related LRR protein 1-like and a Toll/Interleukin-1 receptor-like (TIR)-NBS-LRR, that were upregulated by 34% and 60%, respectively, in resistant P. massoniana. PRM verification confirmed the overexpression of LRR proteins, indicating their potential role in resistance to B. xylophilus. In agreement with this view, the downregulation of CC-NBS-LRR in vulnerable strains of P. massoniana is involved in the pathogenic pathways of nematode infestation [23]. Serine/arginine-rich (SR) proteins are the key players of alternative splicing, emerging as a critical co-transcriptional regulation for plants in response to environmental stresses [24]. The protein level of this splicing factor RSZ21A increased 44% in our resistant P. massoniana strains. This regulation seems to be common in plants and humans in response to pathogen or stress [25, 26].

PLAs (phospholipase A) function to release fatty acids from membrane lipids. In general, phospholipase genes are beneficial for crop yield due to an associated increase in resistance to a variety of pathogen infections and abiotic stresses (reviewed in [27]). Here, a huge increase in PLA1-Iγ3 (>5-fold) was observed in the resistant P. massoniana. PLAI in Arabidopsis (AtPLAI), an acyl hydrolase that is different from other specific phospholipase A group members, is involved in basal jasmonic acid (JA) production for resistance against the necrotrophic fungus B. cinerea [28]. It is likely that JA production in our resistant Masson pine contributes highly to this resistance. Another important, and the most upregulated, protein in the resistant P. massoniana is a putative peroxisomal biogenesis factor (peroxin). Peroxisomes are small, membrane-enclosed organelles that contain enzymes involved in a variety of biochemical pathways in different types of cells. In plants, fatty acid oxidation is restricted to peroxisomes, which makes them a major source of metabolic energy. Increased peroxin might be needed to produce more peroxisomes. Thus, enhanced and/or induced peroxisome synthesis may trigger grave consequences for cell fate such as malignant degeneration, but may also rescue cells or tissues from the damaging effects of such radicals [29]. Thus, the enhanced peroxisomes would benefit P. massoninato fight B. xylophilus infestation and to provide metabolites in a variety of physiological processes.

Terpene synthases were often found to contribute to defense against B. xylophilus in P. massoniana [4, 6, 13] and another Pinus Spp. [15]. In this study, the expression of a few monoterpene synthases was found increased, but not significantly to meet the criteria of the greater-than 30% cutoff, possibly due to no infestation before sampling.

Taken together, these findings suggest that the highly resistant P. Massoniana might possess a few mechanisms for resistance to B. xylophilus infection and even to other unrelated pathogens. Detailed characterization of the molecular signals that specify the expression of resistance may lead to novel strategies for the protection of P. massoniana against PWD.

Analysis of the resistance network using the downregulated proteins

Interestingly, within the 96 proteins with reduced expression, there were four isoperoxidases (POXs; a decrease of 51%, 33%, 33%, and 23%, respectively) and two laccases (a decrease of 54% and 32%). POXs and laccases are frequently associated with plant defense, which is enhanced after a pathogen attack. Physiologically, POXs also play diverse important roles in the plant life cycle, such as in cell wall metabolism, lignification, suberization, and ROS metabolism. The reason why the seedlings of resistant clones expressed significantly lower levels of peroxidases and laccases than susceptible strains without infestation is elusive. Presumably, these reduced POXs act as an alternative enhanced defense mechanism after pathogen attacks as observed in another study [13] for greater production of phenolic compounds, including synthesis of quinones, tannins, and melanins, and for the polymerization of lignin and suberin. Tannins and melanins are toxic to pathogens, and lignin and suberin are involved in structural defenses. This induction strategy is often used by many species (reviewed in [30]). Another explanation could be the high level of H₂O₂ in the resistant P. massoniana as a passive defense, as per the previously reported negative correlation between the levels of peroxidases and H₂O₂ in the special zone of zucchini (Cucurbita pepo) [31]. However, more superoxide radical (O₂⁻) and H₂O₂ accumulated in the susceptible Masson pine after inoculation with PWN and maintained constant under no PWN challenge condition [13]. The definition of the importance of individual POXs could be difficult because of their low substrate specificity in vitro and the presence of many isoenzymes. The activity and protein level quantification of each POX after induction of injury by infestation, hurt, or other stresses would provide some hints.

Xyloglucan endotransglucosylase/hydrolase (XTHs; EC 2.4.1.207 and/or EC 3.2.1.151), a xyloglucan-modifying enzyme, has been proposed to have an important role during fruit ripening in the decomposition of polymers of the cell wall [32]. Pectic and hemicellulosic polysaccharides, two of the major cell wall components, would not be solubilized and depolymerized if XTHs expression were reduced. In this study, we found that XTHs expression was lessened by 33% in resistant strains compared to vulnerable strains. And three pectinesterases decreased by 46%, 27%, and 26%. Moreover, one pectin acetylesterase (PAEs) decrease by 63% and one putative pectin methylesterase (fragment) (PME), by 43%. Some galacturonyl residues in pectin are often O-acetylated, which may lift the digestibility of pectin. Therefore, a reduction in PAEs likely protects pine from infestation by B. xylophilus. PAEs are widespread in the plant pathogenic oomycetes [33] that consume host pectin as a carbon source. Similarly, demethylesterified pectin may undergo depolymerization by glycosidases. A reduction in PME indicates the protective potential for resistant P. massoniana against pathogens. A direct correlation between wall PME activity and strawberry tissue softening has been established [34]. Interestingly, phytopathogenic fungi and bacteria also produce PME for providing virulence to hosts [35].

Additionally, other proteins such as two lipases and one lipid-binding protein, oxidative stress 3 (putative isoform 1), two L-ascorbate oxidases, Class IV chitinase Chia4-Pa1, thaumatin-like protein L2, and a few glycosyltransferases were all expressed significantly less in resistant strains than in susceptible strains. The results are exceptionally unexpected because these gene products are mostly resistance genes in different species [36–40]. As discussed earlier, the activation of genetic and metabolic defenses of plants when facing pathogenic invaders could be gained. These low-expressed proteins would be induced when facing pathogenic attack [38], probably in case of B. xylophilus attack on P. massoniana. This occurs in Norway spruce (Picea abies) after inoculation with Heterobasidion annosum, and this infection has been demonstrated to massively and quickly upregulate chitinase IV to reduce or prevent pathogen colonization [41]. A similar result indicates that the Chia4‐Pa gene is predominantly expressed for resistance in the single-cell zone surrounding the corrosion cavity in Norway spruce seeds [42].

Collectively, the bred highly resistant P. massoniana has, very likely, harbored multiple common pathways, both passive and active, to defend against B. xylophilus infestation. These findings suggest that Masson pine may be resistant not only to B. xylophilus, but also to more unrelated pathogens. Characterization of the molecular signals for intercellular and intracellular barriers as quantitative trait loci (known as QTLs) that specify the expression of resistant genes may lead to more reliable strategies for plant disease control [43].

Though these results are preliminary, they are very intriguing, since the data were collected from highly resistant clones after around 20 years of breeding and field testing. Some more proteins such as low PSII accumulation 1 (chloroplastic isoform X1, encoded by LPA1, increase >5-fold), adenosylhomocysteinase (increase by 60%), sucrose synthase (increase by 52%), ferredoxin (reduction to 27.8%), and abscisic acid stress ripening protein homolog (reduction to 46%) are also very interesting (S3 Table). LPA1 appears to be an integral membrane chaperone that is required for efficient PSII assembly, probably through direct interaction with the PSII reaction center protein D1 [44]. P. massoniana might own a defense system against infestation when combined with efficient PSII biogenesis and maintenance, which has enormous economic potential. On the other hand, susceptible clones can be planted in non-infested zones in order to maintain plant community diversity.

Supporting information

S1 Table. The protein numbers of GO functional analysis with up-regulation in resistant seedlings.

(DOCX)

Click here for additional data file.^{(16.3KB, docx)}

S2 Table. The protein numbers of GO functional analysis with down-regulation in resistant seedlings.

(DOCX)

Click here for additional data file.^{(16.9KB, docx)}

S3 Table. List of the up-regulated and down-regulated proteins in resistant vs. susceptible strains.

(DOCX)

Click here for additional data file.^{(33.7KB, docx)}

Acknowledgments

The authors greatly thank Drs. Kuanyu Li and Fengmao Cheng for his suggestions during the writing of this manuscript.

Data Availability

The mass spectrometry proteomics data were deposited to the ProteomeXchange (http://proteomecentral.proteomexchange.org/cgi/GetDataset) consortium through the PRIDE partner repository with the dataset identifier PXD030664.

Funding Statement

Xu L.[T000522] Fifth Batch of Anhui Province Innovation and Entrepreneurship Leading Talent Special Support Program http://www.ahxf.gov.cn/ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Gao J. [2020dsgzs16] Science Education and Research Project of Education Department of Anhui Province http://jyt.ah.gov.cn/ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.De Guiran G, Bruguier N. Hybridization and Phylogeny of the Pine Wood Nematode (b uRsaphelenchus Spp.). Nematologica. 1989;35(3):321–30. doi: 10.1163/002825989x00421 [DOI] [Google Scholar]
2.Li Y, Dong Z, Chen D, Zhao S, Zhou F, Cao X, et al. Growth decline of Pinus Massoniana in response to warming induced drought and increasing intrinsic water use efficiency in humid subtropical China. Dendrochronologia. 2019;57:125609. doi: 10.1016/j.dendro.2019.125609 [DOI] [Google Scholar]
3.Liu G, Lin X, Xu S, Liu G, Liu Z, Liu F, et al. Efficacy of fluopyram as a candidate trunk-injection agent against Bursaphelenchus xylophilus. Eur J Plant Pathol. 2020;157(2):403–11. doi: 10.1007/s10658-020-02023-8 [DOI] [Google Scholar]
4.Liu B, Liu Q, Zhou Z, Yin H, Xie Y, Wei Y. Two terpene synthases in resistant Pinus massoniana contribute to defence against Bursaphelenchus xylophilus. Plant, Cell & Environment. 2020;44(1):257–74. doi: 10.1111/pce.13873 [DOI] [PubMed] [Google Scholar]
5.Nose M, Shiraishi S. Breeding for Resistance to Pine Wilt Disease. 2008:334–50. doi: 10.1007/978-4-431-75655-2_34 [DOI] [Google Scholar]
6.Li R, Chen P, Zhu L, Wu F, Chen Y, Zhu P, et al. Characterization and Function of the 1-Deoxy-D-xylose-5-Phosphate Synthase (DXS) Gene Related to Terpenoid Synthesis in Pinus massoniana. International Journal of Molecular Sciences. 2021;22(2):848. doi: 10.3390/ijms22020848 [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Yan X, Cheng X-Y, Wang Y-S, Luo J, Mao Z-C, Ferris VR, et al. Comparative transcriptomics of two pathogenic pinewood nematodes yields insights into parasitic adaptation to life on pine hosts. Gene. 2012;505(1):81–90. doi: 10.1016/j.gene.2012.05.041 [DOI] [PubMed] [Google Scholar]
8.Donald PA, Stamps WT, Linit MJ. Pine wilt. The Plant Health Instructor. 2003. doi: 10.1094/phi-i-2003-0130-01 [DOI] [Google Scholar]
9.Zhang W, Wang X, Li Y, Liu Z, Li D, Wen X, et al. Pinewood Nematode Alters the Endophytic and Rhizospheric Microbial Communities of Pinus massoniana. Microb Ecol. 2021;81(3):807–17. doi: 10.1007/s00248-020-01619-1 . [DOI] [PubMed] [Google Scholar]
10.Ponpandian LN, Rim SO, Shanmugam G, Jeon J, Park YH, Lee SK, et al. Phylogenetic characterization of bacterial endophytes from four Pinus species and their nematicidal activity against the pine wood nematode. Sci Rep. 2019;9(1):12457. doi: 10.1038/s41598-019-48745-6 ; PubMed Central PMCID: PMC6713757. [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Park AR, Jeong SI, Jeon HW, Kim J, Kim N, Ha MT, et al. A Diketopiperazine, Cyclo-(L-Pro-L-Ile), Derived From Bacillus thuringiensis JCK-1233 Controls Pine Wilt Disease by Elicitation of Moderate Hypersensitive Reaction. Front Plant Sci. 2020;11:1023. doi: 10.3389/fpls.2020.01023 ; PubMed Central PMCID: PMC7396504. [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Carrasquinho I, Lisboa A, Inácio ML, Gonçalves E. Genetic variation in susceptibility to pine wilt disease of maritime pine (Pinus pinaster Aiton) half-sib families. Annals of Forest Science. 2018;75(3). doi: 10.1007/s13595-018-0759-x [DOI] [Google Scholar]
13.Liu Q, Wei Y, Xu L, Hao Y, Chen X, Zhou Z. Transcriptomic Profiling Reveals Differentially Expressed Genes Associated with Pine Wood Nematode Resistance in Masson Pine (Pinus massoniana Lamb.). Scientific Reports. 2017;7(1). doi: 10.1038/s41598-017-04944-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Monaghan J, Zipfel C. Plant pattern recognition receptor complexes at the plasma membrane. Current Opinion in Plant Biology. 2012;15(4):349–57. doi: 10.1016/j.pbi.2012.05.006 [DOI] [PubMed] [Google Scholar]
15.Gaspar D, Trindade C, Usie A, Meireles B, Fortes AM, Guimaraes JB, et al. Comparative Transcriptomic Response of Two Pinus Species to Infection with the Pine Wood Nematode Bursaphelenchus xylophilus. Forests. 2020;11(2). ARTN 204. doi: 10.3390/f11020204 WOS:000519236600114. [DOI] [Google Scholar]
16.Hu LJ, Wu XQ, Ding XL, Ye JR. Comparative transcriptomic analysis of candidate effectors to explore the infection and survival strategy of Bursaphelenchus xylophilus during different interaction stages with pine trees. Bmc Plant Biol. 2021;21(1). ARTN 224. WOS:000652204300001. doi: 10.1186/s12870-021-02993-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Gao J, Xi Q, Sun Z, Toda T. Screening and Breeding of Pinus massoniana Seedlings for Resistance to Pine Wood Nematode. China Forestry Science and Technology. 2009;23(1):91–5. (in Chinese). [Google Scholar]
18.Xu L-Y, Gao J-B, Zhang J, Chen X-L, Jiang C-W. Evaluation of disease resistance of clones of Pinus massoniana to Bursaphelenchus xylophilus. Journal of Anhui Agricultural University. 2011;38(6):848–53. (in Chinese). [Google Scholar]
19.Eckardt NA, Innes R. Resistance rodeo: rounding up the full complement of Arabidopsis NBS-LRR genes. Plant Cell. 2003;15(4):806–7. doi: 10.1105/tpc.150470 ; PubMed Central PMCID: PMC526037. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Jones JDG, Dangl JL. The plant immune system. Nature. 2006;444(7117):323–9. doi: 10.1038/nature05286 [DOI] [PubMed] [Google Scholar]
21.Xia Y, Suzuki H, Borevitz J, Blount J, Guo Z, Patel K, et al. An extracellular aspartic protease functions in Arabidopsis disease resistance signaling. EMBO J. 2004;23(4):980–8. doi: 10.1038/sj.emboj.7600086 ; PubMed Central PMCID: PMC380998. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Dangl JL, Jones JD. Plant pathogens and integrated defence responses to infection. Nature. 2001;411(6839):826–33. doi: 10.1038/35081161 . [DOI] [PubMed] [Google Scholar]
23.Xie WF, Liang GH, Huang AZ, Zhang FP, Guo WS. Comparative study on the mRNA expression of Pinus massoniana infected by Bursaphelenchus xylophilus. J Forestry Res. 2020;31(1):75–86. doi: 10.1007/s11676-018-0824-1 WOS:000511930900007. [DOI] [Google Scholar]
24.Li Y, Guo Q, Liu P, Huang J, Zhang S, Yang G, et al. Dual roles of the serine/arginine-rich splicing factor SR45a in promoting and interacting with nuclear cap-binding complex to modulate the salt-stress response in Arabidopsis. New Phytol. 2021;230(2):641–55. doi: 10.1111/nph.17175 . [DOI] [PubMed] [Google Scholar]
25.Wang Z, Liu Q, Lu J, Fan P, Xie W, Qiu W, et al. Serine/Arginine-rich Splicing Factor 2 Modulates Herpes Simplex Virus Type 1 Replication via Regulating Viral Gene Transcriptional Activity and Pre-mRNA Splicing. J Biol Chem. 2016;291(51):26377–87. doi: 10.1074/jbc.M116.753046 ; PubMed Central PMCID: PMC5159499. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Yu B, Li J, Koh J, Dufresne C, Yang N, Qi S, et al. Quantitative proteomics and phosphoproteomics of sugar beet monosomic addition line M14 in response to salt stress. J Proteomics. 2016;143:286–97. doi: 10.1016/j.jprot.2016.04.011 . [DOI] [PubMed] [Google Scholar]
27.Wang G, Ryu S, Wang X. Plant phospholipases: an overview. Methods Mol Biol. 2012;861:123–37. doi: 10.1007/978-1-61779-600-5_8 . [DOI] [PubMed] [Google Scholar]
28.Yang W, Devaiah SP, Pan X, Isaac G, Welti R, Wang X. AtPLAI is an acyl hydrolase involved in basal jasmonic acid production and Arabidopsis resistance to Botrytis cinerea. J Biol Chem. 2007;282(25):18116–28. doi: 10.1074/jbc.M700405200 . [DOI] [PubMed] [Google Scholar]
29.Schrader M, Fahimi HD. Peroxisomes and oxidative stress. Biochim Biophys Acta. 2006;1763(12):1755–66. doi: 10.1016/j.bbamcr.2006.09.006 . [DOI] [PubMed] [Google Scholar]
30.Hiraga S, Sasaki K, Ito H, Ohashi Y, Matsui H. A large family of class III plant peroxidases. Plant Cell Physiol. 2001;42(5):462–8. doi: 10.1093/pcp/pce061 . [DOI] [PubMed] [Google Scholar]
31.Dunand C, De Meyer M, Crèvecoeur M, Penel C. Expression of a peroxidase gene in zucchini in relation with hypocotyl growth. Plant Physiology and Biochemistry. 2003;41(9):805–11. doi: 10.1016/s0981-9428(03)00125-6 [DOI] [Google Scholar]
32.Munoz-Bertomeu J, Miedes E, Lorences EP. Expression of xyloglucan endotransglucosylase/hydrolase (XTH) genes and XET activity in ethylene treated apple and tomato fruits. J Plant Physiol. 2013;170(13):1194–201. doi: 10.1016/j.jplph.2013.03.015 . [DOI] [PubMed] [Google Scholar]
33.Kong GH, Wan L, Deng YZ, Yang WS, Li W, Jiang LQ, et al. Pectin acetylesterase PAE5 is associated with the virulence of plant pathogenic oomycete Peronophythora litchii. Physiol Mol Plant P. 2019;106:16–22. doi: 10.1016/j.pmpp.2018.11.006 WOS:000466356200003. [DOI] [Google Scholar]
34.Draye M, Van Cutsem P. Pectin methylesterases induce an abrupt increase of acidic pectin during strawberry fruit ripening. J Plant Physiol. 2008;165(11):1152–60. doi: 10.1016/j.jplph.2007.10.006 . [DOI] [PubMed] [Google Scholar]
35.Giovane A, Servillo L, Balestrieri C, Raiola A, D’Avino R, Tamburrini M, et al. Pectin methylesterase inhibitor. Biochim Biophys Acta. 2004;1696(2):245–52. doi: 10.1016/j.bbapap.2003.08.011 . [DOI] [PubMed] [Google Scholar]
36.Ben-Amar A, Allel D, Mliki A. Up-regulation of a stress-responsive endochitinase VvChit-IV in grapevine cell cultures improves in vitro stress tolerance. Protoplasma. 2022. Epub 20220104. doi: 10.1007/s00709-021-01733-y . [DOI] [PubMed] [Google Scholar]
37.He L, Li L, Zhu Y, Pan Y, Zhang X, Han X, et al. BolTLP1, a Thaumatin-like Protein Gene, Confers Tolerance to Salt and Drought Stresses in Broccoli (Brassica oleracea L. var. Italica). Int J Mol Sci. 2021;22(20). Epub 20211015. doi: 10.3390/ijms222011132 ; PubMed Central PMCID: PMC8537552. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Lee H-J, Park OK. Lipases associated with plant defense against pathogens. Plant Science. 2019;279:51–8. doi: 10.1016/j.plantsci.2018.07.003 [DOI] [PubMed] [Google Scholar]
39.Pan Y, Wen S, Chen X, Gao X, Zeng X, Liu X, et al. UDP-glycosyltransferases contribute to spirotetramat resistance in Aphis gossypii Glover. Pestic Biochem Physiol. 2020;166:104565. Epub 20200323. doi: 10.1016/j.pestbp.2020.104565 . [DOI] [PubMed] [Google Scholar]
40.Singh RR, Nobleza N, Demeestere K, Kyndt T. Ascorbate Oxidase Induces Systemic Resistance in Sugar Beet Against Cyst Nematode Heterodera schachtii. Front Plant Sci. 2020;11:591715. Epub 20201022. doi: 10.3389/fpls.2020.591715 ; PubMed Central PMCID: PMC7641898. [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Hietala AM, Kvaalen H, Schmidt A, Jøhnk N, Solheim H, Fossdal CG. Temporal and Spatial Profiles of Chitinase Expression by Norway Spruce in Response to Bark Colonization by Heterobasidion annosum. Applied and Environmental Microbiology. 2004;70(7):3948–53. doi: 10.1128/AEM.70.7.3948-3953.2004 [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Wiweger M, Farbos I, Ingouff M, Lagercrantz U, Von Arnold S. Expression of Chia4-Pa chitinase genes during somatic and zygotic embryo development in Norway spruce (Picea abies): similarities and differences between gymnosperm and angiosperm class IV chitinases. J Exp Bot. 2003;54(393):2691–9. doi: 10.1093/jxb/erg299 . [DOI] [PubMed] [Google Scholar]
43.Staskawicz BJ, Ausubel FM, Baker BJ, Ellis JG, Jones JD. Molecular genetics of plant disease resistance. Science. 1995;268(5211):661–7. doi: 10.1126/science.7732374 . [DOI] [PubMed] [Google Scholar]
44.Peng L, Ma J, Chi W, Guo J, Zhu S, Lu Q, et al. LOW PSII ACCUMULATION1 is involved in efficient assembly of photosystem II in Arabidopsis thaliana. Plant Cell. 2006;18(4):955–69. doi: 10.1105/tpc.105.037689 ; PubMed Central PMCID: PMC1425854. [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0273010.r001

Decision Letter 0

Jon M Jacobs

29 May 2022

PONE-D-22-06507Proteomic analysis of Masson pine with high resistance to pine wood nematodesPLOS ONE

Dear Dr. Xu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jul 13 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Jon M. Jacobs, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors present a proteomics study in two groups of Masson pine samples with contrasting response to PWN. The used biological material came from individuals selected and tested for 20 years regarding their resistance to PWN. Several putatively important proteins associated to resistance are identified and discussed. This is an important topic given the damaging potential of PWD on pine forests worldwide and the results presented are relevant for PWD research.

However, several major issues should be taken into consideration by the authors to improve the manuscript. These include (1) a more detailed description of the biological materials used and sampling, (2) clarification of the PWD resistance evaluation procedures of the Masson pine clones, and a (3) more focused comparative discussion of the results with previously published work on PWD resistance in pines, as follows:

(1) It is not clear why were 9 lines (what is the definition of a line?) selected, does this mean that each biological sample is a mixture of 3 individual trees? Are these 9 lines from the same or different families? Was the PWN inoculum prepared from several isolates? How were the symptoms evaluated? How long after inoculation was the sampling performed?

(2) The results from the inoculation and the evaluation of resistance are not quantitatively described. If this has not been previously published (in which case a reference should be provided) further detail is needed.

(3) the results from differentially expressed proteins identified should be discussed within the context of previously published results, either using similar comparative approaches, either proteomic or transcriptomic, within the same pine species between resistant and susceptible individuals, e.g. P. pinaster. Such discussion can help to clarify if resistance mechanisms may be similar or specific.

In addition to these major concerns, additional comments are listed below:

Abstract - Please rephrase “Most of the downregulated proteins seemed to take back seats prior to pathogen attacks”

Introduction – the sentence “The endophytic and rhizospheric microbial community

changes that are potentially caused by B. xylophilus infection of pines [9]” seems to be truncated

Results and Discussion – Fig. 1. What is meant by field vaccination?

I could not have access to the data by following the provided link. A supplementary table with detailed info about the proteins and their classification in GO terms (Fig 3) is missing.

The authors claim they found 42 resistant genes that were upregulated in resistant

P. massoniana compared to vulnerable clones. What do the authors mean by resistance genes? Did they use any available database such as Plant Resistance Genes database (Osuna-Cruz et al., 2017)?

It is mentioned that “Though SA was not detected in resistant P. massoniana lines here, the highly expressed aspartic proteases suggest a stronger defense system.” Stronger that what? Please clarify.

Please rephrase the sentence “Thus, tremendously enhanced peroxisomes

would benefit P. massonina as an “evil” to fight B. xylophilus infestation and as a “good” to provide metabolites in a variety of physiological processes.”

Reviewer #2: This manuscript reports on the analysis of proteomic changes in Pinus massoniana resistant and susceptible plants after inoculation with pinewood nematode, the causal agent of pine wilt disease. This manuscript is relevant to understanding better the mechanisms of P. massoniana resistance to this parasite and can be interesting for researchers working on pine wilt disease. However, the manuscript needs improvement before it can be accepted for publishing, especially in the discussion and conclusions.

Throughout the manuscript it is stated that P. massoniana resistant plants “evolved” certain mechanisms of resistance. However, pinewood nematode was introduced recently (40 years ago) in China, giving no time for a long lived tree to evolve resistance to the disease. Instead, resistance is likely to occur due to natural standing variation, and has probably evolved though other selective pressures or genetic drift. Please remove the references to evolution throughout the manuscript.

Introduction:

Line 44: The first time Monochamus alternatus is mentioned there is no explanation why it is important for pine wilt disease, which is confusing. It should be mentioned before that it is the insect vector.

Lines 72-75: Why give details about P. yunnanensis resistance when there is a paper about P. massoniana resistance? Should this not be the focus?

Methods:

In the Methods, more details should be given. Were there any controls inoculated with water? What is/are the sampling timepoint/s? What is the comparison made to obtained differential expression results? Resistant vs control? Resistant vs susceptible? When it says up- or downregulated, what is compared to what? If the comparison is between susceptible and resistant it would be clearer to talk about proteins more expressed in one or the other.

Lines 101-103: This is not very clear. Were the samples pooled in groups of 3 for protein extraction? Needs to be rephrased.

Line 107: Why use only the phloem?

Lines 163-165: This sentence needs more details. “multiple repetitions of each sample” – How many repetitions? And what part of the protocol was repeated?; “average values between the two samples” – What two samples?; “differential expression of the control group” – What control group?

Lines 165-171: What is the program and version used for the statistical analysis?

Results and discussion:

Discussion is confusing in the way it is presented. Why not compare the proteomics results with transcriptomics available for several species after pinewood nematode inoculation, and specifically for P. massoniana resistant varieties (Liu et al. 2017, Scientific Reports)?

Lines 175-179: There should be some results presented here about the inoculation assays, like percentage of plants that are resistant, the differences between families, the disease progression during the time of the experiments, etc. Has this part of the work never been published before? If it was, please provide reference(s).

Fig. 1: “vaccination”?

Lines 215-216: There is no table or image representing expression levels of the proteins. I suggest it is added, even if it is as supplemental material.

Fig. 3: The font is too small, it is difficult to read. (C) What is log2 fold enrichment? Is it a GO enrichment analysis? please make the legend of the image more clear

Lines 238-239: What are resistant genes? Do you mean candidate genes for resistance?

Lines 252-254: This sentence is not clear. Is it a comparison with results obtained elsewhere?

Lines 254-258: I do not see the connection of resistance genes/receptors to alternative splicing? The discussion about the possible role of alternative splicing in resistance is also not clear.

Lines 275-276: It is not clear what you want to say here.

Lines 292-294: This is not supported by what was observed in Liu et al. (2017, Scientific Reports), in which values of H2O2 were similar between resistant and susceptible P. massoniana before pinewood nematode inoculation.

Lines 307-310: Susceptibility to what?

Lines 316-319: This is the opposite of what was found in some transcriptomics papers of pine response to pinewood nematode. This difference should be discussed.

Lines 319-326: I do not understand this part of the discussion and how it relates to your results.

Conclusions:

The text is confusing and it seems like the continuation of the discussion, instead of conclusions.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2022 Aug 12;17(8):e0273010. doi: 10.1371/journal.pone.0273010.r002

Author response to Decision Letter 0

5 Jul 2022

Dear editorial staffs,

Thanks to the editor and the two reviewers for intensively reviewing our manuscript. It helps a lot to improve our manuscript. We are happy to be given the opportunity to revise the manuscript. We carefully considered the suggestions or comments and answer the questions. During the revision, we have added more supplemental data to support our claim. The point-to-point responses to the comments were prepared. Please see the below.

Responses to Review #1’s Comments

Answer: Sorry for not clearing the information in the manuscript. We modified some. The 9 lines are 9 individual seedlings from different families. To make it clearer, we changed the word “line” into “strain”. More detail modification or correction sees the manuscript. We quote some here: “PWN KS-3B, the most virulent isolate, was selected from 40 infected dead Masson pine in different locations. After 90 days of inoculation with PWN, the first evaluation was performed in July 2003. Seedlings were inoculated with a suspension in 0.5 mL of water containing a mixed population of 5000 nematodes. P. taeda and P. elliottii were set as the resistant control species. Healthy seedlings from 44,400 seedlings of 384 families were tested. Three to five seedlings were selected from each family as resistant candidates. A total of 1201 Masson pine resistant candidates from 251 families were selected, and these individuals were planted according to a row spacing of 2 × 3 m. In 2006, the shoots from the resistant candidates were grafted and P. elliottii was used as the rootstock. The second and third evaluation were performed for two consecutive years in July 2007 and July 2008. PWN was inoculated with a suspension in 0.5 mL of water containing a mixed population of 10,000 nematodes in the field and virulence was determined if the selected grafted seedlings were resistant each year.

Answer: Yes, we published one paper concerning the inoculation and the evaluation of resistance. Here is the one “Liu-yi Xu, Jing-bin Gao, Jian Zhang, Xue-lian Chen, Chun-wu Jiang. Evaluation of disease resistance of clones of Pinus massoniana to Bursaphelenchus xylophilus. Journal of Anhui Agricultural University. 2011, 38(6): 848-853. (In Chinese).” We added the reference (#18) into the manuscript.

(3) The results from differentially expressed proteins identified should be discussed within the context of previously published results, either using similar comparative approaches, either proteomic or transcriptomic, within the same pine species between resistant and susceptible individuals, e.g. P. pinaster. Such discussion can help to clarify if resistance mechanisms may be similar or specific.

Answer: We added more comparison with more similar works produced using pine species. Please see the tracked manuscript.

In addition to these major concerns, additional comments are listed below:

Abstract - Please rephrase “Most of the downregulated proteins seemed to take back seats prior to pathogen attacks”

Answer: We rephrase as “Most of the downregulated proteins are supposed to act as an alternative mechanism for latter enhancement after pathogen attacks”.

Introduction – the sentence “The endophytic and rhizospheric microbial community changes that are potentially caused by B. xylophilus infection of pines [9]” seems to be truncated.

Answer: Sorry, it should be “The endophytic and rhizospheric microbial community changes are potentially caused by B. xylophilus infection in pines”.

Results and Discussion – Fig. 1. What is meant by field vaccination?

Answer: It means “the phenotype 90 days post field inoculation”. To avoid the misunderstanding, we change the wording as “The phenotype 90 days post field inoculation of B. xylophilus”.

I could not have access to the data by following the provided link. A supplementary table with detailed info about the proteins and their classification in GO terms (Fig 3) is missing.

Answer: We supplement Table S1- S3 for Fig. 3 and 4, both of which together are original Fig. 3. Please check it.

The authors claim they found 42 resistant genes that were upregulated in resistant P. massoniana compared to vulnerable clones. What do the authors mean by resistance genes? Did they use any available database such as Plant Resistance Genes database (Osuna-Cruz et al., 2017)?

Answer: Sorry, we checked http://prgdb.org/prgdb4/, any information about pine wilt disease has not been available. We deleted “resistant".

It is mentioned that “Though SA was not detected in resistant P. massoniana strains here, the highly expressed aspartic proteases suggest a stronger defense system.” Stronger that what? Please clarify.

Answer: Sorry for not clarifying it. Now we correct as “…..the highly expressed aspartic proteases suggest its big contribution to the defense system against PWN.”

Please rephrase the sentence “Thus, tremendously enhanced peroxisomeswould benefit P. massonina as an “evil” to fight B. xylophilus infestation and as a “good” to provide metabolites in a variety of physiological processes.”

Answer: Here is the rephrased one: “Thus, the enhanced peroxisomes would benefit P. massonina to fight B. xylophilus infestation and to provide metabolites in a variety of physiological processes”.

Responses to Review #2’s Comments

Throughout the manuscript it is stated that P. massoniana resistant plants “evolved” certain mechanisms of resistance. However, pinewood nematode was introduced recently (40 years ago) in China, giving no time for a long-lived tree to evolve resistance to the disease. Instead, resistance is likely to occur due to natural standing variation, and has probably evolved though other selective pressures or genetic drift. Please remove the references to evolution throughout the manuscript.

Answer: Many thanks to Reviewer 2 for positive acknowledgment. For the “evolve” using, we changed the wording by using “harbor”, “possess”, or “own”. What we expressed is that Masson pine is resistant not only to B. xylophilus, but also to more unrelated pathogens in general. The specificity of any resistance gene needs to be further investigated.

Introduction:

Answer: Thanks. We modified as “At present, PWD is mainly prevented by the application of quarantine and chemical pesticides to control pine wood nematodes (PWNs; Bursaphelenchus xylophilus) and to kill Monochamus alternatus that is an insect vector”.

Lines 72-75: Why give details about P. yunnanensis resistance when there is a paper about P. massoniana resistance? Should this not be the focus?

Answer: We referred to it in Introduction to introduce the background in the field.

Methods:

Answer: Now we gave more detail information as suggested. It seems reviewer #2 misunderstood the materials we described in the manuscript. Sorry for not clarifying well. We did not use water as control, but species P. taeda and P. elliottii were used as positive resistant controls. We screened three times in 2003, 2007, and 2008, respectively. The final resistant materials were used for this study. For proteomic data, it is common to say “upregulation” or “downregulation”. In principle, the expression levels were compared between resistant and susceptible strains.

Lines 101-103: This is not very clear. Were the samples pooled in groups of 3 for protein extraction? Needs to be rephrased.

Answer: We rephrase as “According to the results of the clone resistance evaluation, the highly resistant single trees from different families were selected as the high-resistance strains, and the nonresistant single trees were used as the susceptible group. Nine strains were randomly selected from each group, and three of them were pooled as one biological sample, with a total of 3 biological replicates”.

Line 107: Why use only the phloem?

Answer: We used the phloem because phloem is the first position PWN entering when Monochamus alternatus brings PWN to Masson pine. Definitely, it would be better if we use both the needle and phloem. Sorry, we missed it here.

Answer: The sampling is actually described in “Biological material, pine wood nematode inoculation, and sampling”. Each group has three biological repeats. Each repeat is a pool of 3 individual seedlings from different families with the similar resistance. We have made it clear in Manuscript. No protocol was repeated. We quote here: “First, the average quantitative value was calculated for the three repeats of each group, the resistant or susceptible. The value from the susceptible group was taken as a control. The ratio of the average values between the two groups was calculated and taken as the final differential expression levels. The log2 calculation was performed to gain the relative quantitative values, which conforms to a normal distribution. The two-tailed t-test method was used to calculate the p-value. A change of differential expression exceeding 1.3 was considered as a significant upregulation when the p-value <0.05, while a change less than 0.7 was considered as a significant downregulation when the p-value <0.05”.

Lines 165-171: What is the program and version used for the statistical analysis?

Answer: Office Excel 2016.

Results and discussion:

Answer: Sorry for not comparing more with comparable data in the literature because there are no many data feasible for comparison. Though, we compared more with the paper reviewer#2 mentioned in Results and Discussion (Liu, 2017 ref#13). I quote two here “Similar results were obtained in a genomic transcriptional study, in which genes encoding pectate lyase were found upregulated in the resistant Masson pine compared with the susceptible clones (Liu Q,2017(ref.12).” and “Terpene synthases were often found to contribute to defense against B. xylophilus in P. massoniana (ref. #4,6,13) and another Pinus Spp. (ref#15). In this study, the expression of a few monoterpene synthases was found increased, but not significantly to meet the criteria of the greater-than 30% cutoff, possibly due to no infestation before sampling”.

Answer: Yes, a part of the work was published as “Jingbin Gao, Qijun Xi, Zhuyi Sun, TadaoToda. Screening and Breeding of Pinus massoniana Seedlings for Resistant to Pine Wood Nematode. Development of forestry science and technology. 2009, 23(1), 91-95 (ref#17, published in Chinese)”. We referred to it in the manuscript.

Fig. 1: “vaccination”?

Answer: Here is the change in Fig. 1B: “The phenotype 90 days post field inoculation of B. xylophilus” as we answered reviewer #1.

Lines 215-216: There is no table or image representing expression levels of the proteins. I suggest it is added, even if it is as supplemental material.

Answer: All the tables are presented as supplemental data in the revision.

Fig. 3: The font is too small, it is difficult to read. (C) What is log2 fold enrichment? Is it a GO enrichment analysis? please make the legend of the image more clear

Answer: Sorry, the font is really too small. Figure 3 is split into Figure 3 (original Figure 3A and B) and Figure 4 (original Figure 3C) in order to enlarge the font. The current Figure 4 is a GO enrichment analysis. Now we modified the legend and made it clearer.

Lines 238-239: What are resistant genes? Do you mean candidate genes for resistance?

Answer: It was not accurate. Now the “resistant” was deleted in the Revision.

Lines 252-254: This sentence is not clear. Is it a comparison with results obtained elsewhere?

Answer: Now it is corrected as “In agreement with this view, the downregulation of CC-NBS-LRR in vulnerable strains of P. massoniana is involved in the pathogenic pathways of nematode infestation [ref#23, Xie, 2020]”.

Lines 254-258: I do not see the connection of resistance genes/receptors to alternative splicing? The discussion about the possible role of alternative splicing in resistance is also not clear.

Answer: We did not clearly express our data that the protein levels of serine/arginine-rich splicing factor RSZ21A increased 44% in our resistant Masson pine. Here is the correction: “Serine/arginine-rich (SR) proteins are the key players of alternative splicing, emerging as a critical co-transcriptional regulation for plants in response to environmental stresses [ref#24 Li, 2021]. The protein level of this splicing factor RSZ21A increased 44% in our resistant P. massoniana strains. This regulation seems to be common in plants and humans in response to pathogen or stress [ref#25&26, Wang,2016; Yu,2016]”.

Lines 275-276: It is not clear what you want to say here.

Answer: Here is the correction: “Taken together, these findings suggest that the highly resistant P. massoniana might possess a few mechanisms for resistance to B. xylophilus infection and even to other unrelated pathogens”.

Answer: Yes, we added this information in the Revision. Here is the quotation: “However, superoxide radical (O2−) and H2O2 accumulated more in the susceptible Masson pine after inoculation with PWN, but maintained constant under no PWN-challenge condition (ref#13, Liu, 2017)”. We also cite the paper (ref#13Liu, 2017) concerning the expression of POX as “Presumably, these reduced POXs act as an alternative enhanced defense mechanism after pathogen attacks as observed in another study (ref#13Liu, 2017) for greater production of phenolic compounds…..”.

Lines 307-310: Susceptibility to what?

Answer: Sorry, we deleted.

Lines 316-319: This is the opposite of what was found in some transcriptomics papers of pine response to pinewood nematode. This difference should be discussed.

Answer: Thanks for reminding. We add more references concerning the opposite results. Here is the quotation: “The results are exceptionally unexpected because these gene products are mostly resistance genes in different species (ref#36-40: PMID: 30709493, 33193547, 34984633, 34681789, 32448419)”.

Lines 319-326: I do not understand this part of the discussion and how it relates to your results.

Answer: The results from original Lines 316-319 are different from other transcriptomic data as reviewer#2 commented. Lines 319-326 are the assumption we proposed and cited some supported evidence.

Conclusions:

The text is confusing and it seems like the continuation of the discussion, instead of conclusions.

Answer: We agree and delete “Conclusion” in Revision.

Thanks to both reviewers and the editor for your comments on our manuscript. We have revised all by point-to-point responses. Hopefully, it fulfills the criteria for publication.

Sincerely,

Jingbin Gao

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(27.8KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0273010.r003

Decision Letter 1

Jon M Jacobs

1 Aug 2022

Proteomic analysis of Masson pine with high resistance to pine wood nematodes

PONE-D-22-06507R1

Dear Dr. Xu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Jon M. Jacobs, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0273010.r004

Acceptance letter

Jon M Jacobs

4 Aug 2022

PONE-D-22-06507R1

Proteomic analysis of Masson pine with high resistance to pine wood nematodes

Dear Dr. Xu:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr Jon M. Jacobs

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. The protein numbers of GO functional analysis with up-regulation in resistant seedlings.

(DOCX)

Click here for additional data file.^{(16.3KB, docx)}

S2 Table. The protein numbers of GO functional analysis with down-regulation in resistant seedlings.

(DOCX)

Click here for additional data file.^{(16.9KB, docx)}

S3 Table. List of the up-regulated and down-regulated proteins in resistant vs. susceptible strains.

(DOCX)

Click here for additional data file.^{(33.7KB, docx)}

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(27.8KB, docx)}

Data Availability Statement

[pone.0273010.ref001] 1.De Guiran G, Bruguier N. Hybridization and Phylogeny of the Pine Wood Nematode (b uRsaphelenchus Spp.). Nematologica. 1989;35(3):321–30. doi: 10.1163/002825989x00421 [DOI] [Google Scholar]

[pone.0273010.ref002] 2.Li Y, Dong Z, Chen D, Zhao S, Zhou F, Cao X, et al. Growth decline of Pinus Massoniana in response to warming induced drought and increasing intrinsic water use efficiency in humid subtropical China. Dendrochronologia. 2019;57:125609. doi: 10.1016/j.dendro.2019.125609 [DOI] [Google Scholar]

[pone.0273010.ref003] 3.Liu G, Lin X, Xu S, Liu G, Liu Z, Liu F, et al. Efficacy of fluopyram as a candidate trunk-injection agent against Bursaphelenchus xylophilus. Eur J Plant Pathol. 2020;157(2):403–11. doi: 10.1007/s10658-020-02023-8 [DOI] [Google Scholar]

[pone.0273010.ref004] 4.Liu B, Liu Q, Zhou Z, Yin H, Xie Y, Wei Y. Two terpene synthases in resistant Pinus massoniana contribute to defence against Bursaphelenchus xylophilus. Plant, Cell & Environment. 2020;44(1):257–74. doi: 10.1111/pce.13873 [DOI] [PubMed] [Google Scholar]

[pone.0273010.ref005] 5.Nose M, Shiraishi S. Breeding for Resistance to Pine Wilt Disease. 2008:334–50. doi: 10.1007/978-4-431-75655-2_34 [DOI] [Google Scholar]

[pone.0273010.ref006] 6.Li R, Chen P, Zhu L, Wu F, Chen Y, Zhu P, et al. Characterization and Function of the 1-Deoxy-D-xylose-5-Phosphate Synthase (DXS) Gene Related to Terpenoid Synthesis in Pinus massoniana. International Journal of Molecular Sciences. 2021;22(2):848. doi: 10.3390/ijms22020848 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0273010.ref007] 7.Yan X, Cheng X-Y, Wang Y-S, Luo J, Mao Z-C, Ferris VR, et al. Comparative transcriptomics of two pathogenic pinewood nematodes yields insights into parasitic adaptation to life on pine hosts. Gene. 2012;505(1):81–90. doi: 10.1016/j.gene.2012.05.041 [DOI] [PubMed] [Google Scholar]

[pone.0273010.ref008] 8.Donald PA, Stamps WT, Linit MJ. Pine wilt. The Plant Health Instructor. 2003. doi: 10.1094/phi-i-2003-0130-01 [DOI] [Google Scholar]

[pone.0273010.ref009] 9.Zhang W, Wang X, Li Y, Liu Z, Li D, Wen X, et al. Pinewood Nematode Alters the Endophytic and Rhizospheric Microbial Communities of Pinus massoniana. Microb Ecol. 2021;81(3):807–17. doi: 10.1007/s00248-020-01619-1 . [DOI] [PubMed] [Google Scholar]

[pone.0273010.ref010] 10.Ponpandian LN, Rim SO, Shanmugam G, Jeon J, Park YH, Lee SK, et al. Phylogenetic characterization of bacterial endophytes from four Pinus species and their nematicidal activity against the pine wood nematode. Sci Rep. 2019;9(1):12457. doi: 10.1038/s41598-019-48745-6 ; PubMed Central PMCID: PMC6713757. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0273010.ref011] 11.Park AR, Jeong SI, Jeon HW, Kim J, Kim N, Ha MT, et al. A Diketopiperazine, Cyclo-(L-Pro-L-Ile), Derived From Bacillus thuringiensis JCK-1233 Controls Pine Wilt Disease by Elicitation of Moderate Hypersensitive Reaction. Front Plant Sci. 2020;11:1023. doi: 10.3389/fpls.2020.01023 ; PubMed Central PMCID: PMC7396504. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0273010.ref012] 12.Carrasquinho I, Lisboa A, Inácio ML, Gonçalves E. Genetic variation in susceptibility to pine wilt disease of maritime pine (Pinus pinaster Aiton) half-sib families. Annals of Forest Science. 2018;75(3). doi: 10.1007/s13595-018-0759-x [DOI] [Google Scholar]

[pone.0273010.ref013] 13.Liu Q, Wei Y, Xu L, Hao Y, Chen X, Zhou Z. Transcriptomic Profiling Reveals Differentially Expressed Genes Associated with Pine Wood Nematode Resistance in Masson Pine (Pinus massoniana Lamb.). Scientific Reports. 2017;7(1). doi: 10.1038/s41598-017-04944-7 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0273010.ref014] 14.Monaghan J, Zipfel C. Plant pattern recognition receptor complexes at the plasma membrane. Current Opinion in Plant Biology. 2012;15(4):349–57. doi: 10.1016/j.pbi.2012.05.006 [DOI] [PubMed] [Google Scholar]

[pone.0273010.ref015] 15.Gaspar D, Trindade C, Usie A, Meireles B, Fortes AM, Guimaraes JB, et al. Comparative Transcriptomic Response of Two Pinus Species to Infection with the Pine Wood Nematode Bursaphelenchus xylophilus. Forests. 2020;11(2). ARTN 204. doi: 10.3390/f11020204 WOS:000519236600114. [DOI] [Google Scholar]

[pone.0273010.ref016] 16.Hu LJ, Wu XQ, Ding XL, Ye JR. Comparative transcriptomic analysis of candidate effectors to explore the infection and survival strategy of Bursaphelenchus xylophilus during different interaction stages with pine trees. Bmc Plant Biol. 2021;21(1). ARTN 224. WOS:000652204300001. doi: 10.1186/s12870-021-02993-9 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0273010.ref017] 17.Gao J, Xi Q, Sun Z, Toda T. Screening and Breeding of Pinus massoniana Seedlings for Resistance to Pine Wood Nematode. China Forestry Science and Technology. 2009;23(1):91–5. (in Chinese). [Google Scholar]

[pone.0273010.ref018] 18.Xu L-Y, Gao J-B, Zhang J, Chen X-L, Jiang C-W. Evaluation of disease resistance of clones of Pinus massoniana to Bursaphelenchus xylophilus. Journal of Anhui Agricultural University. 2011;38(6):848–53. (in Chinese). [Google Scholar]

[pone.0273010.ref019] 19.Eckardt NA, Innes R. Resistance rodeo: rounding up the full complement of Arabidopsis NBS-LRR genes. Plant Cell. 2003;15(4):806–7. doi: 10.1105/tpc.150470 ; PubMed Central PMCID: PMC526037. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0273010.ref020] 20.Jones JDG, Dangl JL. The plant immune system. Nature. 2006;444(7117):323–9. doi: 10.1038/nature05286 [DOI] [PubMed] [Google Scholar]

[pone.0273010.ref021] 21.Xia Y, Suzuki H, Borevitz J, Blount J, Guo Z, Patel K, et al. An extracellular aspartic protease functions in Arabidopsis disease resistance signaling. EMBO J. 2004;23(4):980–8. doi: 10.1038/sj.emboj.7600086 ; PubMed Central PMCID: PMC380998. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0273010.ref022] 22.Dangl JL, Jones JD. Plant pathogens and integrated defence responses to infection. Nature. 2001;411(6839):826–33. doi: 10.1038/35081161 . [DOI] [PubMed] [Google Scholar]

[pone.0273010.ref023] 23.Xie WF, Liang GH, Huang AZ, Zhang FP, Guo WS. Comparative study on the mRNA expression of Pinus massoniana infected by Bursaphelenchus xylophilus. J Forestry Res. 2020;31(1):75–86. doi: 10.1007/s11676-018-0824-1 WOS:000511930900007. [DOI] [Google Scholar]

[pone.0273010.ref024] 24.Li Y, Guo Q, Liu P, Huang J, Zhang S, Yang G, et al. Dual roles of the serine/arginine-rich splicing factor SR45a in promoting and interacting with nuclear cap-binding complex to modulate the salt-stress response in Arabidopsis. New Phytol. 2021;230(2):641–55. doi: 10.1111/nph.17175 . [DOI] [PubMed] [Google Scholar]

[pone.0273010.ref025] 25.Wang Z, Liu Q, Lu J, Fan P, Xie W, Qiu W, et al. Serine/Arginine-rich Splicing Factor 2 Modulates Herpes Simplex Virus Type 1 Replication via Regulating Viral Gene Transcriptional Activity and Pre-mRNA Splicing. J Biol Chem. 2016;291(51):26377–87. doi: 10.1074/jbc.M116.753046 ; PubMed Central PMCID: PMC5159499. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0273010.ref026] 26.Yu B, Li J, Koh J, Dufresne C, Yang N, Qi S, et al. Quantitative proteomics and phosphoproteomics of sugar beet monosomic addition line M14 in response to salt stress. J Proteomics. 2016;143:286–97. doi: 10.1016/j.jprot.2016.04.011 . [DOI] [PubMed] [Google Scholar]

[pone.0273010.ref027] 27.Wang G, Ryu S, Wang X. Plant phospholipases: an overview. Methods Mol Biol. 2012;861:123–37. doi: 10.1007/978-1-61779-600-5_8 . [DOI] [PubMed] [Google Scholar]

[pone.0273010.ref028] 28.Yang W, Devaiah SP, Pan X, Isaac G, Welti R, Wang X. AtPLAI is an acyl hydrolase involved in basal jasmonic acid production and Arabidopsis resistance to Botrytis cinerea. J Biol Chem. 2007;282(25):18116–28. doi: 10.1074/jbc.M700405200 . [DOI] [PubMed] [Google Scholar]

[pone.0273010.ref029] 29.Schrader M, Fahimi HD. Peroxisomes and oxidative stress. Biochim Biophys Acta. 2006;1763(12):1755–66. doi: 10.1016/j.bbamcr.2006.09.006 . [DOI] [PubMed] [Google Scholar]

[pone.0273010.ref030] 30.Hiraga S, Sasaki K, Ito H, Ohashi Y, Matsui H. A large family of class III plant peroxidases. Plant Cell Physiol. 2001;42(5):462–8. doi: 10.1093/pcp/pce061 . [DOI] [PubMed] [Google Scholar]

[pone.0273010.ref031] 31.Dunand C, De Meyer M, Crèvecoeur M, Penel C. Expression of a peroxidase gene in zucchini in relation with hypocotyl growth. Plant Physiology and Biochemistry. 2003;41(9):805–11. doi: 10.1016/s0981-9428(03)00125-6 [DOI] [Google Scholar]

[pone.0273010.ref032] 32.Munoz-Bertomeu J, Miedes E, Lorences EP. Expression of xyloglucan endotransglucosylase/hydrolase (XTH) genes and XET activity in ethylene treated apple and tomato fruits. J Plant Physiol. 2013;170(13):1194–201. doi: 10.1016/j.jplph.2013.03.015 . [DOI] [PubMed] [Google Scholar]

[pone.0273010.ref033] 33.Kong GH, Wan L, Deng YZ, Yang WS, Li W, Jiang LQ, et al. Pectin acetylesterase PAE5 is associated with the virulence of plant pathogenic oomycete Peronophythora litchii. Physiol Mol Plant P. 2019;106:16–22. doi: 10.1016/j.pmpp.2018.11.006 WOS:000466356200003. [DOI] [Google Scholar]

[pone.0273010.ref034] 34.Draye M, Van Cutsem P. Pectin methylesterases induce an abrupt increase of acidic pectin during strawberry fruit ripening. J Plant Physiol. 2008;165(11):1152–60. doi: 10.1016/j.jplph.2007.10.006 . [DOI] [PubMed] [Google Scholar]

[pone.0273010.ref035] 35.Giovane A, Servillo L, Balestrieri C, Raiola A, D’Avino R, Tamburrini M, et al. Pectin methylesterase inhibitor. Biochim Biophys Acta. 2004;1696(2):245–52. doi: 10.1016/j.bbapap.2003.08.011 . [DOI] [PubMed] [Google Scholar]

[pone.0273010.ref036] 36.Ben-Amar A, Allel D, Mliki A. Up-regulation of a stress-responsive endochitinase VvChit-IV in grapevine cell cultures improves in vitro stress tolerance. Protoplasma. 2022. Epub 20220104. doi: 10.1007/s00709-021-01733-y . [DOI] [PubMed] [Google Scholar]

[pone.0273010.ref037] 37.He L, Li L, Zhu Y, Pan Y, Zhang X, Han X, et al. BolTLP1, a Thaumatin-like Protein Gene, Confers Tolerance to Salt and Drought Stresses in Broccoli (Brassica oleracea L. var. Italica). Int J Mol Sci. 2021;22(20). Epub 20211015. doi: 10.3390/ijms222011132 ; PubMed Central PMCID: PMC8537552. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0273010.ref038] 38.Lee H-J, Park OK. Lipases associated with plant defense against pathogens. Plant Science. 2019;279:51–8. doi: 10.1016/j.plantsci.2018.07.003 [DOI] [PubMed] [Google Scholar]

[pone.0273010.ref039] 39.Pan Y, Wen S, Chen X, Gao X, Zeng X, Liu X, et al. UDP-glycosyltransferases contribute to spirotetramat resistance in Aphis gossypii Glover. Pestic Biochem Physiol. 2020;166:104565. Epub 20200323. doi: 10.1016/j.pestbp.2020.104565 . [DOI] [PubMed] [Google Scholar]

[pone.0273010.ref040] 40.Singh RR, Nobleza N, Demeestere K, Kyndt T. Ascorbate Oxidase Induces Systemic Resistance in Sugar Beet Against Cyst Nematode Heterodera schachtii. Front Plant Sci. 2020;11:591715. Epub 20201022. doi: 10.3389/fpls.2020.591715 ; PubMed Central PMCID: PMC7641898. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0273010.ref041] 41.Hietala AM, Kvaalen H, Schmidt A, Jøhnk N, Solheim H, Fossdal CG. Temporal and Spatial Profiles of Chitinase Expression by Norway Spruce in Response to Bark Colonization by Heterobasidion annosum. Applied and Environmental Microbiology. 2004;70(7):3948–53. doi: 10.1128/AEM.70.7.3948-3953.2004 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0273010.ref042] 42.Wiweger M, Farbos I, Ingouff M, Lagercrantz U, Von Arnold S. Expression of Chia4-Pa chitinase genes during somatic and zygotic embryo development in Norway spruce (Picea abies): similarities and differences between gymnosperm and angiosperm class IV chitinases. J Exp Bot. 2003;54(393):2691–9. doi: 10.1093/jxb/erg299 . [DOI] [PubMed] [Google Scholar]

[pone.0273010.ref043] 43.Staskawicz BJ, Ausubel FM, Baker BJ, Ellis JG, Jones JD. Molecular genetics of plant disease resistance. Science. 1995;268(5211):661–7. doi: 10.1126/science.7732374 . [DOI] [PubMed] [Google Scholar]

[pone.0273010.ref044] 44.Peng L, Ma J, Chi W, Guo J, Zhu S, Lu Q, et al. LOW PSII ACCUMULATION1 is involved in efficient assembly of photosystem II in Arabidopsis thaliana. Plant Cell. 2006;18(4):955–69. doi: 10.1105/tpc.105.037689 ; PubMed Central PMCID: PMC1425854. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Proteomic analysis of Masson pine with high resistance to pine wood nematodes

Jingbin GAO

Ting PAN

Xuelian CHEN

Qiang Wei

Liuyi Xu

Roles

Abstract

Introduction

Materials and methods

Biological material, pine wood nematode inoculation, and sampling

Tandem mass tag (TMT) quantitative proteome analysis

Sample preparation

Protein extraction and trypsin digestion

TMT labeling

HPLC fractionation

LC–MS/MS analysis

Database search and bioinformatics analysis

Quantitative verification of targeted proteome by mass spectrometry-based parallel reaction monitoring (PRM)

LC–MS analysis

Proteomic statistical analysis of the data

Results and discussion

Evaluation of the resistance of Masson pine clones to PWD

Fig 1. Resistance screening of Masson pine seedlings 90 days after inoculation with PWN.

General proteomic analysis

Fig 2. Repeatability of the samples and overall distribution diagram of the differentially expressed proteins.

Functional classification of the differentially expressed proteins

Fig 3. Distribution of the differentially expressed proteins in the GO secondary annotations and GO-directed acyclic graph in resistant versus susceptible seedlings.

Fig 4. GO enrichment showing the functions of the differentially expressed proteins.

Analysis of the resistance network using the upregulated proteins

Analysis of the resistance network using the downregulated proteins

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Jon M Jacobs

Roles

Author response to Decision Letter 0

Decision Letter 1

Jon M Jacobs

Roles

Acceptance letter

Jon M Jacobs

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases