The Bursaphelenchus xylophilus Effector BxNMP1 Targets PtTLP-L2 to Mediate PtGLU Promoting Parasitism and Virulence in Pinus thunbergii.

Pinus is an important economic tree species, but pine wilt disease (PWD) seriously threatens the survival of pine trees. PWD caused by Bursaphelenchus xylophilus is a major quarantine disease worldwide that causes significant economic losses. However, more information about its molecular pathogenesis is needed, resulting in a lack of effective prevention and treatment measures. In recent years, effectors have become a hot topic in exploring the molecular pathogenic mechanism of pathogens. Here, we identified a specific effector, BxNMP1, from B. xylophilus. In situ hybridization experiments revealed that BxNMP1 was specifically expressed in dorsal gland cells and intestinal cells, and RT-qPCR experiments revealed that BxNMP1 was upregulated in the early stage of infection. The sequence of BxNMP1 was different in the avirulent strain, and when BxNMP1-silenced B. xylophilus was inoculated into P. thunbergii seedlings, the disease severity significantly decreased. We demonstrated that BxNMP1 interacted with the thaumatin-like protein PtTLP-L2 in P. thunbergii. Additionally, we found that the ß-1,3-glucanase PtGLU interacted with PtTLP-L2. Therefore, we hypothesized that BxNMP1 might indirectly interact with PtGLU through PtTLP-L2 as an intermediate mediator. Both targets can respond to infection, and PtTLP-L2 can enhance the resistance of pine trees. Moreover, we detected increased salicylic acid contents in P. thunbergii seedlings inoculated with B. xylophilus when BxNMP1 was silenced or when the PtTLP-L2 recombinant protein was added. In summary, we identified a key virulence effector of PWNs, BxNMP1. It positively regulates the pathogenicity of B. xylophilus and interacts directly with PtTLP-L2 and indirectly with PtGLU. It also inhibits the expression of two targets and the host salicylic acid pathway. This study provides theoretical guidance and a practical basis for controlling PWD and breeding for disease resistance.

Effective Synthesis of High-Integrity mRNA Using In Vitro Transcription.

mRNA vaccines are entering a period of rapid development. However, their synthesis is still plagued by challenges related to mRNA impurities and fragments (incomplete mRNA). Most impurities of mRNA products transcribed in vitro are mRNA fragments. Only full-length mRNA transcripts containing both a 5'-cap and a 3'-poly(A) structure are viable for in vivo expression. Therefore, RNA fragments are the primary product-related impurities that significantly hinder mRNA efficacy and must be effectively controlled; these species are believed to originate from either mRNA hydrolysis or premature transcriptional termination. In the manufacturing of commercial mRNA vaccines, T7 RNA polymerase-catalyzed in vitro transcription (IVT) synthesis is a well-established method for synthesizing long RNA transcripts. This study identified a pivotal domain on the T7 RNA polymerase that is associated with erroneous mRNA release. By leveraging the advantageous properties of a T7 RNA polymerase mutant and precisely optimized IVT process parameters, we successfully achieved an mRNA integrity exceeding 91%, thereby further unlocking the immense potential of mRNA therapeutics.

The Growth-Promoting and Colonization of the Pine Endophytic Pseudomonas abietaniphila for Pine Wilt Disease Control.

In this study, we focused on evaluating the impact of Pseudomonas abietaniphila BHJ04 on the growth of Pinus massoniana seedlings and its biocontrol efficacy against pine wilt disease (PWD). Additionally, the colonization dynamics of P. abietaniphila BHJ04 on P. massoniana were examined. The growth promotion experiment showed that P. abietaniphila BHJ04 significantly promoted the growth of the branches and roots of P. massoniana. Pot control experiments indicated that strain BHJ04 significantly inhibited the spread of PWD. There were significant changes in the expression of several genes related to pine wood nematode defense in P. massoniana, including chitinase, nicotinamide synthetase, and triangular tetrapeptide-like superfamily protein isoform 9. Furthermore, our results revealed significant upregulation of genes associated with the water stress response (dehydration-responsive proteins), genetic material replication (DNA/RNA polymerase superfamily proteins), cell wall hydrolase, and detoxification (cytochrome P450 and cytochrome P450 monooxygenase superfamily genes) in the self-regulation of P. massoniana. Colonization experiments demonstrated that strain BHJ04 can colonize the roots, shoots, and leaves of P. massoniana, and the colonization amount on the leaves was the greatest, reaching 160,000 on the 15th day. However, colonization of the stems lasted longer, with the highest level of colonization observed after 45 d. This study provides a preliminary exploration of the growth-promoting and disease-preventing mechanisms of P. abietaniphila BHJ04 and its ability to colonize pines, thus providing a new biocontrol microbial resource for the biological control of plant diseases.

A pioneer nematode effector suppresses plant reactive oxygen species burst by interacting with the class III peroxidase.

Bursaphelenchus xylophilus is the pathogen of pine wilt disease, which can devastate the pine forest ecosystem. Usually, plant cells generate reactive oxygen species (ROS) as a defensive substance or signalling molecules to resist the infection of nematodes. However, little is known about how B. xylophilus effectors mediate the plant ROS metabolism. Here, we identified a pioneer B. xylophilus Prx3-interacting effector 1 (BxPIE1) expressed in the dorsal gland cells and the intestine. Silencing of the BxPIE1 gene resulted in reduced nematode reproduction and a delay in disease progression during parasitic stages, with the upregulation of pathogenesis-related (PR) genes PtPR-3 (class Ⅳ chitinase) and PtPR-9 (peroxidase). The protein-protein interaction assays further demonstrated that BxPIE1 interacts with a Pinus thunbergii class III peroxidase (PtPrx3), which produces H2O2 under biotic stress. The expression of BxPIE1 and PtPrx3 was upregulated during the infection stage. Furthermore, BxPIE1 effectively inhibited H2O2 generating from class III peroxidase and ascorbate can recover the virulence of siBxPIE1-treated B. xylophilus by scavenging H2O2. Taken together, BxPIE1 is an important virulence factor, revealing a novel mechanism utilized by nematodes to suppress plant immunity.

Pinus thunbergii Parl. Somatic Plants' Resistance to Bursaphelenchus xylophilus Depends on Pathogen-Induced Differential Transcriptomic Responses.

Pinus thunbergii Parl. is an economically and medicinally important plant, as well as a world-renowned horticultural species of the Pinus genus. Pine wilt disease is a dangerous condition that affects P. thunbergii. However, understanding of the genetics underlying resistance to this disease is poor. Our findings reveal that P. thunbergii's resistance mechanism is based on differential transcriptome responses generated by the early presence of the pathogen Bursaphelenchus xylophilus, also known as the pine wood nematode. A transcriptome analysis (RNA-seq) was performed to examine gene expression in shoot tissues from resistant and susceptible P. thunbergii trees. RNA samples were collected from the shoots of inoculated pines throughout the infection phases by the virulent Bursaphelenchus xylophilus AMA3 strain. The photosynthesis and plant-pathogen interaction pathways were significantly enriched in the first and third days after infection. Flavonoid biosynthesis was induced in response to late infestation (7 and 14 days post-infestation). Calmodulin, RBOH, HLC protein, RPS, PR1, and genes implicated in phytohormone crosstalk (e.g., SGT1, MYC2, PP2C, and ERF1) showed significant alterations between resistant and susceptible trees. Furthermore, salicylic acid was found to aid pine wood nematodes tolerate adverse conditions and boost reproduction, which may be significant for pine wood nematode colonization within pines. These findings provide new insights into how host defenses overcame pine wood nematode infection in the early stage, which could potentially contribute to the development of novel strategies for the control of pine wilt disease.

Heat stress-induced oviposition behavioral change correlates with sperm damage in the pine sawyer beetle, Monochamus alternatus.

BACKGROUND: Global climate change is causing an increase in extreme high temperatures (EHTs), which subject insects to unprecedented stress. Behavior plasticity in response to EHTs, particularly oviposition behavior, is important for the persistence and outbreak of insect populations. Investigating the plasticity of oviposition behavior and its underlying mechanisms has theoretical importance to pest management, but knowledge gaps still remain. RESULTS: Herein, we characterized the reproductive traits of Monochamus alternatus, a dominant insect vector of the destructive pine wilt disease, including oviposition behavioral patterns, fecundity, offspring fitness and sperm viability, under simulated heatwave conditions in the laboratory. The results showed that (i) EHTs induced a novel oviposition behavior, whereby females deposited multiple eggs into a single groove rather than laying one egg per groove under normal condition; (ii) EHTs exerted stage- and sex-specific effects on fecundity, offspring fitness and sperm viability; and (iii) there was a significant correlation between frequency of the novel oviposition strategy and sperm viability. CONCLUSION: We hypothesized that this beetle pest has the ability to flexibly shift towards a low-cost oviposition strategy to counteract the fitness costs caused by heat stress. Taken together, these findings provide a theoretical foundation for personalized pest management strategies in the context of climate change. © 2024 Society of Chemical Industry.

Effective synthesis of circRNA via a thermostable T7 RNA polymerase variant as the catalyst.

Introduction: Circular RNAs (circRNAs) are endogenous noncoding RNAs (ncRNAs) with transcriptional lengths ranging from hundreds to thousands. circRNAs have attracted attention owing to their stable structure and ability to treat complicated diseases. Our objective was to create a one-step reaction for circRNA synthesis using wild-type T7 RNA polymerase as the catalyst. However, T7 RNA polymerase is thermally unstable, and we streamlined circRNA synthesis via consensus and folding free energy calculations for hotspot selection. Because of the thermal instability, the permuted intron and exon (PIE) method for circRNA synthesis is conducted via tandem catalysis with a transcription reaction at a low temperature and linear RNA precursor cyclization at a high temperature. Methods: To streamline the process, a multisite mutant T7 RNA polymerase (S430P, N433T, S633P, F849I, F880Y, and G788A) with significantly improved thermostability was constructed, and G788A was used. Results: The resulting mutant exhibited stable activity at 45°C for over an hour, enabling the implementation of a one-pot transcription and cyclization reaction. The simplified circRNA production process demonstrated an efficiency comparable to that of the conventional two-step reaction, with a cyclization rate exceeding 95% and reduced production of immunostimulatory dsRNA byproducts.

Mechanism of Salt Tolerance and Plant Growth Promotion in Priestia megaterium ZS-3 Revealed by Cellular Metabolism and Whole-Genome Studies.

Approximately one-third of agricultural land worldwide is affected by salinity, which limits the productivity and sustainability of crop ecosystems. Plant-growth-promoting rhizobacteria (PGPR) are a potential solution to this problem, as PGPR increases crop yield through improving soil fertility and stress resistance. Previous studies have shown that Priestia megaterium ZS-3(ZS-3) can effectively help plants tolerate salinity stress. However, how ZS-3 regulates its metabolic adaptations in saline environments remains unclear. In this study, we monitored the metabolic rearrangement of compatibilisers in ZS-3 and combined the findings with genomic data to reveal how ZS-3 survives in stressful environments, induces plant growth, and tolerates stress. The results showed that ZS-3 tolerated salinity levels up to 9%. In addition, glutamate and trehalose help ZS-3 adapt to osmotic stress under low NaCl stress, whereas proline, K+, and extracellular polysaccharides regulate the osmotic responses of ZS-3 exposed to high salt stress. Potting experiments showed that applying the ZS-3 strain in saline and neutral soils could effectively increase the activities of soil acid phosphatase, urease, and invertase in both soils, thus improving soil fertility and promoting plant growth. In addition, strain ZS-3-GFP colonised the rhizosphere and leaves of Cinnamomum camphora well, as confirmed by confocal microscopy and resistance plate count analysis. Genomic studies and in vitro experiments have shown that ZS-3 exhibits a variety of beneficial traits, including plant-promoting, antagonistic, and other related traits (such as resistance to saline and heavy metal stress/tolerance, amino acid synthesis and transport, volatile compound synthesis, micronutrient utilisation, and phytohormone biosynthesis/regulatory potential). The results support that ZS-3 can induce plant tolerance to abiotic stresses. These data provide important clues to further reveal the interactions between plants and microbiomes, as well as the mechanisms by which micro-organisms control plant health.

Resistant and Susceptible Pinus thunbergii ParL. Show Highly Divergent Patterns of Differentially Expressed Genes during the Process of Infection by Bursaphelenchus xylophilus.

Pine wilt disease (PWD) is a devastating disease that threatens pine forests worldwide, and breeding resistant pines is an important management strategy used to reduce its impact. A batch of resistant seeds of P. thunbergii was introduced from Japan. Based on the resistant materials, we obtained somatic plants through somatic embryogenesis. In this study, we performed transcriptome analysis to further understand the defense response of resistant somatic plants of P. thunbergii to PWD. The results showed that, after pine wood nematode (PWN) infection, resistant P. thunbergii stimulated more differential expression genes (DEGs) and involved more regulatory pathways than did susceptible P. thunbergii. For the first time, the alpha-linolenic acid metabolism and linoleic acid metabolism were intensively observed in pines resisting PWN infection. The related genes disease resistance protein RPS2 (SUMM2) and pathogenesis-related genes (PR1), as well as reactive oxygen species (ROS)-related genes were significantly up-expressed in order to contribute to protection against PWN inoculation in P. thunbergii. In addition, the diterpenoid biosynthesis pathway was significantly enriched only in resistant P. thunbergii. These findings provided valuable genetic information for future breeding of resistant conifers, and could contribute to the development of new diagnostic tools for early screening of resistant pine seedlings based on specific PWN-tolerance-related markers.

The Bursaphelenchus xylophilus candidate effector BxLip-3 targets the class I chitinases to suppress immunity in pine.

Lipase is involved in lipid hydrolysis, which is related to nematodes' energy reserves and stress resistance. However, the role of lipases in Bursaphelenchus xylophilus, a notorious plant-parasitic nematode responsible for severe damage to pine forest ecosystems, remains largely obscure. Here, we characterized a class III lipase as a candidate effector and named it BxLip-3. It was transcriptionally up-regulated in the parasitic stages of B. xylophilus and specifically expressed in the oesophageal gland cells and the intestine. In addition, BxLip-3 suppressed cell death triggered by the pathogen-associated molecular patterns PsXEG1 and BxCDP1 in Nicotiana benthamiana, and its Lipase-3 domain is essential for immunosuppression. Silencing of the BxLip-3 gene resulted in a delay in disease onset and increased the activity of antioxidant enzymes and the expression of pathogenesis-related (PR) genes. Plant chitinases are thought to be PR proteins involved in the defence system against pathogen attack. Using yeast two-hybrid and co-immunoprecipitation assays, we identified two class I chitinases in Pinus thunbergii, PtChia1-3 and PtChia1-4, as targets of BxLip-3. The expression of these two chitinases was up-regulated during B. xylophilus inoculation and inhibited by BxLip-3. Overall, this study illustrated that BxLip-3 is a crucial virulence factor that plays a critical role in the interaction between B. xylophilus and host pine.

Optimization of Constitutive Promoters Using a Promoter-Trapping Vector in Burkholderia pyrrocinia JK-SH007.

Selecting suitable promoters to drive gene overexpression can provide significant insight into the development of engineered bacteria. In this study, we analyzed the transcriptome data of Burkholderia pyrrocinia JK-SH007 and identified 54 highly expressed genes. The promoter sequences were located using genome-wide data and scored using the prokaryotic promoter prediction software BPROM to further screen out 18 promoter sequences. We also developed a promoter trap system based on two reporter proteins adapted for promoter optimization in B. pyrrocinia JK-SH007: firefly luciferase encoded by the luciferase gene set (Luc) and trimethoprim (TP)-resistant dihydrofolate reductase (TPr). Ultimately, eight constitutive promoters were successfully inserted into the probe vector and transformed into B. pyrrocinia JK-SH007. The transformants were successfully grown on Tp antibiotic plates, and firefly luciferase expression was determined by measuring the relative light unit (RLU). Five of the promoters (P4, P9, P10, P14, and P19) showed 1.01-2.51-fold higher activity than the control promoter λ phage transcriptional promoter (PRPL). The promoter activity was further validated via qPCR analysis, indicating that promoters P14 and P19 showed stable high transcription levels at all time points. Then, GFP and RFP proteins were overexpressed in JK-SH007. In addition, promoters P14 and P19 were successfully used to drive gene expression in Burkholderia multivorans WS-FJ9 and Escherichia coli S17-1. The two constitutive promoters can be used not only in B. pyrrocinia JK-SH007 itself to gene overexpression but also to expand the scope of application.

Pinus massoniana somatic embryo maturation, mycorrhization of regenerated plantlets and its resistance to Bursaphelenchus xylophilus.

Pine wilt disease, caused by the pine wood nematode (PWN, Bursaphelenchus xylophilus), is a major quarantine forest disease that poses a threat to various pine species, including Pinus massoniana (masson pine), worldwide. Breeding of PWN-resistant pine trees is an important approach to prevent the disease. To expedite the production of PWN-resistant P. massoniana accessions, we investigated the effects of maturation medium treatments on somatic embryo development, germination, survival, and rooting. Furthermore, we evaluated the mycorrhization and nematode resistance of regenerated plantlets. Abscisic acid was identified as the main factor affecting maturation, germination, and rooting of somatic embryos in P. massoniana, resulting in a maximum of 34.9 ± 9.4 somatic embryos per ml, 87.3 ± 9.1% germination rate, and 55.2 ± 29.3% rooting rate. Polyethylene glycol was identified as the main factor affecting the survival rate of somatic embryo plantlets, with a survival rate of up to 59.6 ± 6.8%, followed by abscisic acid. Ectomycorrhizal fungi inoculation with Pisolithus orientalis enhanced the shoot height of plantlets regenerated from embryogenic cell line (ECL) 20-1-7. Ectomycorrhizal fungi inoculation also improved the survival rate of plantlets during the acclimatization stage, with 85% of mycorrhized plantlets surviving four months after acclimatization in the greenhouse, compared with 37% non-mycorrhized plantlets. Following PWN inoculation, the wilting rate and the number of nematodes recovered from ECL 20-1-7 were lower than those recovered from ECL 20-1-4 and 20-1-16. The wilting ratios of mycorrhizal plantlets from all cell lines were significantly lower than those of non-mycorrhizal regenerated plantlets. This plantlet regeneration system and mycorrhization method could be used in the large-scale production of nematode-resistance plantlets and to study the interaction between nematode, pines, and mycorrhizal fungi.

CysB Is a Key Regulator of the Antifungal Activity of Burkholderia pyrrocinia JK-SH007.

Burkholderia pyrrocinia JK-SH007 can effectively control poplar canker caused by pathogenic fungi. Its antifungal mechanism remains to be explored. Here, we characterized the functional role of CysB in B. pyrrocinia JK-SH007. This protein was shown to be responsible for the synthesis of cysteine and the siderophore ornibactin, as well as the antifungal activity of B. pyrrocinia JK-SH007. We found that deletion of the cysB gene reduced the antifungal activity and production of the siderophore ornibactin in B. pyrrocinia JK-SH007. However, supplementation with cysteine largely restored these two abilities in the mutant. Further global transcriptome analysis demonstrated that the amino acid metabolic pathway was significantly affected and that some sRNAs were significantly upregulated and targeted the iron-sulfur metabolic pathway by TargetRNA2 prediction. Therefore, we suggest that, in B. pyrrocinia JK-SH007, CysB can regulate the expression of genes related to Fe-S clusters in the iron-sulfur metabolic pathway to affect the antifungal activity of B. pyrrocinia JK-SH007. These findings provide new insights into the various biological functions regulated by CysB in B. pyrrocinia JK-SH007 and the relationship between iron-sulfur metabolic pathways and fungal inhibitory substances. Additionally, they lay the foundation for further investigation of the main antagonistic substances of B. pyrrocinia JK-SH007.

Transcriptomic Analysis Reveals the Response of the Bacterium Priestia Aryabhattai SK1-7 to Interactions and Dissolution with Potassium Feldspar.

Potassium feldspar (K2O·Al2O3·6SiO2) is considered to be the most important source of potash fertilizer. The use of microorganisms to dissolve potassium feldspar is a low-cost and environmentally friendly method. Priestia aryabhattai SK1-7 is a strain with a strong ability to dissolve potassium feldspar; it showed a faster pH drop and produced more acid in the medium with potassium feldspar as the insoluble potassium source than in the medium with K2HPO4 as the soluble potassium source. We speculated whether the cause of acid production was related to one or more stresses, such as mineral-induced generation of reactive oxygen species (ROS), the presence of aluminum in potassium feldspar, and cell membrane damage due to friction between SK1-7 and potassium feldspar, and analyzed it by transcriptome. The results revealed that the expression of the genes related to pyruvate metabolism, the two-component system, DNA repair, and oxidative stress pathways in strain SK1-7 was significantly upregulated in potassium feldspar medium. The subsequent validation experiments revealed that ROS were the stress faced by strain SK1-7 when interacting with potassium feldspar and led to a decrease in the total fatty acid content of SK1-7. In the face of ROS stress, strain SK1-7 upregulated the expression of the maeA-1 gene, allowing malic enzyme (ME2) to produce more pyruvate to be secreted outside the cell using malate as a substrate. Pyruvate is both a scavenger of external ROS and a gas pedal of dissolved potassium feldspar. IMPORTANCE Mineral-microbe interactions play important roles in the biogeochemical cycling of elements. Manipulating mineral-microbe interactions and optimizing the consequences of such interactions can be used to benefit society. It is necessary to explore the black hole of the mechanism of interaction between the two. In this study, it is revealed that P. aryabhattai SK1-7 faces mineral-induced ROS stress by upregulating a series of antioxidant genes as a passive defense, while overexpression of malic enzyme (ME2) secretes pyruvate to scavenge ROS as well as to increase feldspar dissolution, releasing K, Al, and Si into the medium. Our research provides a theoretical basis for improving the ability of microorganisms to weather minerals through genetic manipulation in the future.

Efficient synthesis of furfurylamine from biomass via a hybrid strategy in an EaCl:Gly-water medium.

The objective of this work was to develop an efficient approach for chemoenzymatically transforming biomass to furfurylamine by bridging chemocatalysis and biocatalysis in a deep eutectic solvent of EaCl:Gly-water. Using hydroxyapatite (HAP) as support, heterogeneous catalyst SO4 2-/SnO2-HAP was synthesized for transforming lignocellulosic biomass into furfural using organic acid as a co-catalyst. The turnover frequency (TOF) was correlated with the pKa value of the used organic acid. Corncob was transformed by oxalic acid (pKa = 1.25) (0.4 wt%) plus SO4 2-/SnO2-HAP (2.0 wt%) to produce furfural with a yield of 48.2% and a TOF of 6.33 h-1 in water. In deep eutectic solvent EaCl:Gly-water (1:2, v/v), co-catalysis with SO4 2-/SnO2-HAP and oxalic acid was utilized to transform corncob, rice straw, reed leaf, and sugarcane bagasse for the production of furfural with the yield of 42.4%-59.3% (based on the xylan content) at 180°C after 10 min. The formed furfural could be efficiently aminated to furfurylamine with E. coli CCZU-XLS160 cells in the presence of NH4Cl (as an amine donor). As a result of the biological amination of furfural derived from corncob, rice straw, reed leaf, and sugarcane bagasse for 24 h, the yields of furfurylamine reached >99%, with a productivity of 0.31-0.43 g furfurylamine per g xylan. In EaCl:Gly-water, an efficient chemoenzymatic catalysis strategy was employed to valorize lignocellulosic biomass into valuable furan chemicals.

Promoting the application of Pinus thunbergii Parl. to enhance the growth and survival rates of post-germination somatic plantlets.

OBJECTIVE: There is a growing need for nematode resistant Pinaceae species plantlets to cope with the global scale degradation of coniferous forests, due to the prevalence of pine wilt disease. One of the bottlenecks that limits the commercialization of Pinaceae species plantlets is regeneration following their transfer from controlled sterile environments to the field while maintaining high survival rates. METHODS: The growth factors of somatic plantlets (SPs), such as sucrose, media, culture substrate, brassinolide and spectrum were investigated to promote the application of somatic nematode-resistant P. thunbergii plants in afforestation. RESULTS: The 1/2 WPM liquid medium, culture substrate (perlite and vermiculite =1:1), and carbohydrate (20 g/L sucrose) were effective in stimulating the growth of rooted SPs. While for unrooted SPs, 1 ug/L of brassinolide enhanced plantlet growth and rooting. And blue light (B) significantly promoted the longitudinal growth of shoots, while red light (R) was beneficial for root growth during the laboratory domestication stage. High quality SPs were obtained at a R/B ratio of 8:2. Following this acclimatization protocol, the P. thunbergii SPs could be directly transplanted to the field with a higher survival rate (85.20 %) in a forcing house. CONCLUSION: this acclimatization protocol extremely improved the survival rate of P. thunbergii SPs. Moreover, this work will contribute to enhancing the possibilities for somatic plant afforestation with Pinus species.

Two Novel Bursaphelenchus xylophilus Kunitz Effector Proteins Using Different Infection and Survival Strategies to Suppress Immunity in Pine.

Pine wilt disease, caused by Bursaphelenchus xylophilus, results in tremendous economic loss in conifer production every year. To disturb the host immune responses, plant pathogens secrete a mass of effector proteins that facilitate the infection process. Although several effectors of B. xylophilus have been identified, detailed mechanisms of their functions remain largely unexplored. Here, we reveal two novel B. xylophilus Kunitz effectors, named BxKU1 and BxKU2, using different infection strategies to suppress immunity in Pinus thunbergii. We found that both BxKU1 and BxKU2 could suppress PsXEG1-triggered cell death and were present in the nucleus and cytoplasm in Nicotiana benthamiana. However, they had different three-dimensional structures and various expression patterns in B. xylophilus infection. In situ hybridization experiments showed that BxKU2 was expressed in the esophageal glands and ovaries, whereas BxKU1 was only expressed in the esophageal glands of females. We further confirmed that the morbidity was significantly decreased in P. thunbergii infected with B. xylophilus when BxKU1 and BxKU2 were silenced. The silenced BxKU2I, but not BxKU1, affected the reproduction and feeding rate of B. xylophilus. Moreover, BxKU1 and BxKU2 targeted to different proteins in P. thunbergii, but they all interacted with thaumatin-like protein 4 (TLP4) according to yeast two-hybrid screening. Collectively, our study showed that B. xylophilus could incorporate two Kunitz effectors in a multilayer strategy to counter immune response in P. thunbergii, which could help us better understand the interaction between plant and B. xylophilus.

Effect of Associated Bacteria GD1 on the Low-Temperature Adaptability of Bursaphelenchus xylophilus Based on RNA-Seq and RNAi.

To explore the effect of associated bacteria on the low-temperature adaptability of pinewood nematodes (PWNs), transcriptome sequencing (RNA-seq) of PWN AH23 treated with the associated bacterial strain Bacillus cereus GD1 was carried out with reference to the whole PWN genome. Bioinformatic software was utilized to analyze the differentially expressed genes (DEGs). This study was based on the analysis of DEGs to verify the function of daf-11 by RNAi. The results showed that there were 439 DEGs between AH23 treated with GD1 and those treated with ddH2O at 10 °C. There were 207 pathways annotated in the KEGG database and 48 terms annotated in the GO database. It was found that after RNAi of daf-11, the survival rate of PWNs decreased significantly at 10 °C, and fecundity decreased significantly at 15 °C. It can be concluded that the associated bacteria GD1 can enhance the expression of genes related to PWN low-temperature adaptation and improve their adaptability to low temperatures.

A real-time PCR for detection of pathogens of anthracnose on Chinese fir using TaqMan probe targeting ApMat gene.

BACKGROUND: Anthracnose is one of the most widespread and destructive diseases on Chinese fir. Colletotrichum cangyuanense, Colletotrichum fructicola, Colletotrichum gloeosporioides, and Colletotrichum siamense are the causal agents of anthracnose on Chinese fir. A rapid and accurate diagnosis of different pathogens is critical for the disease management. RESULTS: Phylogenetic tree and sequence similarity analysis showed that the single-locus ApMat provides superior phylogenetic information and is an appropriate target to distinguish C. cangyuanense, C. fructicola, C. gloeosporioides, and C. siamense. The real-time PCR assays with the primer sets of MQ-F/R, 1#C-F/R, YK-F/R, and WZ-F/R, and corresponding TaqMan probes of MQ-P, 1#C-P, YK-P, and WZ-P were specific for C. cangyuanense, C. fructicola, C. gloeosporioides, and C. siamense, respectively. The sensitivity tests showed that the lowest amount of gDNA that the PCRs can detect was 1 ng of genomic DNA. The validity of the assays was confirmed by directly detecting the pathogens from both the fungal culture and infected Chinese fir. CONCLUSION: These results demonstrated the potential of the TaqMan real-time PCR targeting the ApMat gene to provide rapid, specific, and reliable molecular detection of C. fructicola, C. gloeosporioides, C. siamense, and C. cangyuanense, respectively. The data also provided a reference solution for the identification of species within Colletotrichum gloeosporioides species complex (CGSC), which share similar morphological characteristics. © 2022 Society of Chemical Industry.

Studies on the Requirement of Transthyretin Protein (BxTTR-52) for the Suppression of Host Innate Immunity in Bursaphelenchus xylophilus.

The pinewood nematode, Bursaphelenchus xylophilus, has been determined as one of the world's top ten plant-parasitic nematodes. It causes pine wilt, a progressive disease that affects the economy and ecologically sustainable development in East Asia. B. xylophilus secretes pathogenic proteins into host plant tissues to promote infection. However, little is known about the interaction between B. xylophilus and pines. Previous studies reported transthyretin proteins in some species and their strong correlation with immune evasion, which has also been poorly studied in B. xylophilus. In this study, we cloned and functionally validated the B. xylophilus pathogenic protein BxTTR-52, containing a transthyretin domain. An in situ hybridization assay demonstrated that BxTTR-52 was expressed mainly in the esophageal glands of B. xylophilus. Confocal microscopy revealed that BxTTR-52-RFP localized to the nucleus, cytoplasm, and plasma membrane. BxTTR-52 recombinant proteins produced by Escherichia coli could be suppressed by hydrogen peroxide and antioxidant enzymes in pines. Moreover, silencing BxTTR-52 significantly attenuated the morbidity of Pinus thunbergii infected with B. xylophilus. It also suppressed the expression of pathogenesis-related genes in P. thunbergii. These results suggest that BxTTR-52 suppresses the plant immune response in the host pines and might contribute to the pathogenicity of B. xylophilus in the early infection stages.