The anti-phytoalexin gene Bx-cathepsin W supports the survival of Bursaphelenchus xylophilus under Pinus massoniana phytoalexin stress.

BACKGROUND: Pine trees challenged by Bursaphelenchus xylophilus invasion produce phytoalexins to combat this nematode. Nevertheless, the phytoalexins of Asian pine trees are ineffective against B. xylophilus. The anti-phytoalexin genes of B. xylophilus disable almost all Asian pine phytoalexins, which has allowed B. xylophilus to devastate pine forests in eastern Asia over the last four decades. However, to date, the factors that stimulate anti-phytoalexin gene expression and the mechanisms by which these genes act are not well understood. RESULTS: Here, we described anti-phytoalexin genes in B. xylophilus using transcriptomic and bioinformatics analyses. The genes that were induced by both Pinus massoniana and carvone and had similarly elevated expression trends were considered anti-phytoalexin genes. Altogether, 187 anti-phytoalexin genes were identified, including 4 cathepsin genes. KEGG pathway enrichment indicated that those cathepsins were related to the Lysosome pathway. Since cathepsins help to maintain metabolic homeostasis by participating in the degradation of heterophagic and autophagic material, the lysosomal cathepsin gene Bx-cathepsin W was cloned and characterized. The results of the RNAi assessment indicated that the knockdown of Bx-cathepsin W reduced the survival rates of B. xylophilus under carvone or P. massoniana stress. The correlation between Bx-cathepsin W and the susceptibility of pines showed that Bx-cathepsin W might help improve the anti-phytotoxin ability of B. xylophilus. CONCLUSIONS: The results indicated that the anti-phytoalexin gene Bx-cathepsin W supported the survival of B. xylophilus under P. massoniana phytoalexin stress. The cDNA library sequencing, differentially expressed gene identification, and WGCNA algorithm analysis provided insight at a systemic level into the gene regulation of B. xylophilus in response to the immune reaction of P. massoniana. These results will lead to a better understanding of the function of nematode defenses in host innate immunity.

Trehalose metabolism genes render rice white tip nematode Aphelenchoides besseyi (Nematoda: Aphelenchoididae) resistant to an anaerobic environment.

After experiencing anaerobic environments, Aphelenchoides besseyi will enter a state of suspended animation known as anoxybiosis, during which it may use trehalose as an energy supply to survive. To explore the function of trehalose metabolism, two trehalose-6-phosphate synthase (TPS) genes (Ab-tps1 and Ab-tps2) encoding enzymes catalysing trehalose synthesis, and three trehalase (TRE) genes (Ab-ntre1, Ab-ntre2 and Ab-atre) encoding enzymes catalysing the hydrolysis of trehalose, were identified and investigated. Ab-tps1 and Ab-tps2 were active during certain periods of anoxybiosis for A. besseyi, and Ab-tps2, Ab-ntre1, Ab-ntre2 and Ab-atre were active during certain periods of recovery. The results of RNA interference experiments suggested that TRE genes regulated each other and both TPS genes, while a single TPS gene only regulated the other TPS gene. However, two TPS genes together could regulate TRE genes, which indicated a feedback mechanism between these genes. All these genes also positively regulated the survival and resumption of active metabolism of the nematode. Genes functioning at re-aeration have a greater impact on nematode survival, suggesting that these genes could play roles in anoxybiosis regulation, but may function within restricted time frames. Changes in trehalose levels matched changes in TRE activity during the anoxybiosis-re-aeration process, suggesting that trehalose may act as an energy supply source. The observation of up-regulation of TPS genes during anoxybiosis suggested a possible signal role of trehalose. Trehalose metabolism genes could also work together to control trehalose levels at a certain level when the nematode is under anaerobic conditions.

Trehalose metabolism genes of Aphelenchoides besseyi (Nematoda: Aphelenchoididae) in hypertonic osmotic pressure survival.

Some organisms can survive extreme desiccation caused by hypertonic osmotic pressure by entering a state of suspended animation known as osmobiosis. The free-living mycophagous nematode Aphelenchoides besseyi can be induced to enter osmobiosis by soaking in osmolytes. It is assumed that sugars (in particular trehalose) are instrumental for survival under environmental stress. In A. besseyi, two putative trehalose-6-phosphate synthase genes (TPS) encoding enzymes catalyzing trehalose synthesis, and a putative trehalase gene (TRE) encoding enzymes that catalyze hydrolysis of trehalose were identified and then characterized based on their transcriptome. RT-qPCR analyses showed that each of these genes is expressed as mRNA when A. besseyi is entering in, during and recovering from osmobiosis, but only for certain periods. The changes of TRE activity were consistent with the transcript level changes of the TRE gene, and the trehalose level declined at certain periods when the nematodes were in, as well as recovering from, osmobiosis; this suggested that the hydrolysis of threhalose is essential. The feeding method of RNA interference (RNAi) was used to temporarily knock down the expression of each of the TPS and TRE genes. No obviously different phenotype was observed from any of the genes silenced individually or simultaneously, but the survival under hypertonic osmotic pressure reduced significantly and the recovery was delayed. These results indicated that trehalose metabolism genes should play a role in osmobiosis regulation and function within a restricted time frame.

MicroRNA-mediated susceptible poplar gene expression regulation associated with the infection of virulent Melampsora larici-populina.

BACKGROUND: Rust caused by Melampsora larici-populina is one of the most damaging diseases of poplars. Rust is considered to be a model pathogen for genetic studies because both pathogen and host genomes are available. The poplar 'Robusta', whose general rust resistance is defeated by virulent rust E4, provides suitable host material for studies of the gene regulation involved in rust resistance/susceptibility. In this study, we investigated the microRNA-mediated susceptible poplar gene expression regulation associated with the infection of virulent rust. We were particularly interested in delineating the host-pathogen interactions with a specific focus on microRNAs (miRNAs). RESULTS: To study the susceptibility of poplar to M. larici-populina, small RNA (sRNA) libraries, a degradome cDNA library and digital gene expression libraries were constructed for rust-inoculated and rust-free susceptible poplar 'Robusta' leaves through high-throughput sequencing. Altogether, 12,722 regulating interactions were identified. The results delineated the framework of post-transcriptional regulation of gene expression in the susceptible poplar, which was infected by the virulent rust. The results indicated that pathogen-associated molecular patterns (PAMPs) and PAMP-triggered immunity were induced by the infection of virulent rust E4 and that miRNAs still functioned at this stage. After this stage, miRNA-regulated R genes, such as TIR-NBS-LRR and CC-NBS-LRR, were not fully functional. Additionally, the rust-responsive miRNAs did not regulate the signaling component genes related to the salicylic acid pathway or the hypersensitive response. CONCLUSIONS: We found that the defense-related post-transcriptional regulation of the susceptible poplar 'Robusta' functions normally only at the stage of PAMPs and PAMP-triggered immunity (PTI). More importantly, the miRNA-mediated post-transcriptional regulation of defense signal pathway genes were inactivated by the infection of virulent rust at the stage of effector-triggered susceptibility and during the following stages of salicylic acid and hypersensitive responses. This inactivation was the major characteristic of 'Robusta' susceptibility.