Periodicals：the plant journalif：7.2 (q1)Publication timedoi：10.1111/tpj.16426
Citrus is seriously threatened by huanglongbing (HLB), which not only leads to a decrease in fruit production and poor fruit taste, but also increases the cost of citrus production in affected areas by 40-50%. In addition to starch metabolism disorders, the pathological mechanism of HLB and citrus immune response are still unclear. MiRNA-mediated gene silencing is a negative regulatory defense mechanism, and by regulating the expression of related genes on the defensive behavior of HLB-tolerant citrus varieties, it was found that citrus can sense Albertium asiaticum (CLAS) infection, and endogenous and exogenous methyl salicylate (MESA) can further enhance the induced immune response to HLB.
Citrus trees with mild HLB symptoms were also selected for the experiment, respectively, with CK (100% ethanol. 1 mM Mesa and 1 mM Mesa (100% ethanol solubilized) treated 8 trees, and disease severity was measured every 2 months. The environmental effects of MESA were evaluated by measuring the air MESA emissions of two groups of citrus plants by GC-MS analysis every six months for three consecutive years.
1.Citrus defense response to CLAS infection
A total of about 26 000 annotated citrus transcripts were identified, with a total of 921 significantly differentiated genes, of which 565 were up-regulated and 360 were down-regulated (Fig. 1b). By GO analysis (Figure 2), genes involved in the "redox" process were substantially upregulated upon CLAS infection. Genes involved in the process of "primary metabolic regulation" such as the "auxin reaction" are significantly down-regulated. Among these metabolic processes, "proteolysis", "hydrolase", and "peptidase" are the three most inhibitory growth factors (Figure 2).
Table 1 List of citrus transcripts with significantly differentially expressed expressions
Of these 259 highly expressed citrus genes, only 10 genes showed significant differences in CLAS infection (Figure 1B), of which 8 were down-regulated and 2 were up-regulated (Table 1). Fifteen abundantly expressed differentially expressed genes and 13 randomly expressed mRNAs were screened by RT-PCR experimentally, and the transcripts (CS5G30320 and CS6G07200) showed consistency between RNA-seq and experimental validation, indicating significant reliability of transcriptome results.
Fig.1 Citrus senses CLAS infection and initiates gene reprogramming
Figure 2 Biological processes associated with defense are preferentially activated2.CLAS infection induces the expression of CS1G24440 and the production of MESACS1G24440 (SAMT) is one of two citrus transcripts significantly induced in CLAS infection. The recombinant citrus SAMT expressed in Escherichia coli was also able to convert SA to MESA, suggesting that citrus SAMT is a true methyltransferase. SAMT-OE transgenic citrus is transgenic by fusing SAMT CDS with a 35S promoter. Compared to wild-type plants, SAMT-OE transgenic citrus was significantly elevated in both leaf and canopy space, suggesting that SAMT expression led to more MESA production (Fig. 3D). CLAS-infected citrus released Mesa levels were significantly elevated in canopy space and leaf air, while mock-treated citrus did not release detectable volatile Mesa (Figure 3).
Fig.3 CLAS infection induces the production and emission of methyl salicylate (MESA) in citrus plants3.MESA is a community immune signal that initiates neighboring treesPlant immune-related genes such as pr-2 and pr-3 were significantly induced on days 1 and 3 after MESA treatment (Fig. 4A). To test whether mesa is also effective for the whole plant, spray mesa (01 and 1 mm), and then disease severity checks were performed every 2 months, and after 4 months of MESA treatment, disease symptoms (Figure 4b) and disease severity (Figure 4C) began to decrease compared to the control group.
In order to examine whether elevated MESA in SAMT-OE leaves and canopy space has a positive effect on HLB resistance, a comparative experiment was established in which HLB-infected wild-type citrus coexisted, mixed with uninfected WT or SAMT-OE transgenic citrus in a closed plastic container (Fig. 5A), and the results showed significant differences in PR-2, PR-3, etc., indicating that citrus can redirect the expression of defense-related genes after sensing CLAS (Fig. 5B). After 6 months of co-culture in an enclosed space, it was found that citrus plants showed a stronger tolerant phenotype than wild-type citrus plants compared to disease-resistant symptoms (Figure 5C).
Fig.4 The application of MESA significantly improved the performance of citrus
Fig.5 Expression of defense genes stimulated by canopy MESACitrus plants were studied in closed (top) or open space (bottom) for three consecutive years (2016-2018), and the differences in leaf pathogenic symptoms were more pronounced between the plants preserved in different environments (Fig. 6a). It is hypothesized that the differences between plants can be attributed to differences in MESA in the environment. After measuring the ambient SA concentration every 6 months, the MESA concentration in the enclosed space was higher than in the open area (Figure 6D). Citrus can use MESA as an effective defense mechanism against CLAS infection, limiting disease progression in plants and communities.
Fig.6 MESA significantly improved the disease resistance of citrus4.SAMT expression is regulated at both the transcriptional and post-transcriptional levelsThe SAMT promoter region was analyzed, and 218 transcription factors were identified. To determine whether TF also binds to other citrus genes and whether the expression of these binding genes is induced by CLAS infection. Finally, 14 transcription factors were identified, of which 5 transcription factors were associated with upregulated genes. These transcription factors belong to B3 DNA-binding proteins, among others.
MIR2977 was identified, which is highly inversely complementary to the second exon of SAMT. Expression of MIR2977 increased by 68% after CLAS infection, while expression of SAMT increased approximately 40-fold (Table 1). In MIR2977-OE plants, SAMT expression was significantly reduced compared to wild-type citrus. Under normal conditions, the expression of SAMT is regulated by MIR2977, but when citrus is infected with CLAS, MIR2977 releases inhibition and induces SAMT expression in infected citrus.
5.Citrus miRNAs are key orchestrators of citrus and CLAS interactionsDe-miRNAs did respond to different expressions of differentially expressed genes at the time of CLAS infection, and 791 miRNA pairs were identified, of which 751 mi mRNA pairs with specific gene annotation information were identified for further study, and it was found that most biological processes could be divided into "response to stimulus", "carbohydrate metabolism", and "ion transport" biological processes (Figure 7), and miRNAs were the key regulators of differential expression of citrus genes after CLAS infection.
Fig.7 Comparison of transcriptome and miRNAome analyses reveals key HLB response biological processes
Fig.8 Clas-induced genes promote the expression of genes related to cell death and defense
Citrus miRNAs play a vital role in the plant's immune response. Through the study and analysis of the defensive behavior of HLB-tolerant citrus varieties, it was found that citrus can sense the infection of Free Bacillus asiaticus (CLAS), induce an immune response to HLB, and produce methyl salicylate (MESA). It is proposed that miRNA and related components should be a key target for the engineering or breeding of resistant citrus varieties and rootstocks. MESA is a natural community immune signal, and HLB-infected citrus produce and release more MESA, alerting neighboring citrus plants.
References. cheng b, et al. small rnas contribute to citrus huanglongbing tolerance by manipulating methyl salicylate signaling and exogenous methyl salicylate primes citrus groves from emerging infection. plant j. 2023
One of the authors of this client article is Ling Enshengxin's "big guy" - Zeng Liang
He joined BGI in 2010 and served as the head of bioinformatics, senior bioinformatics analysis engineer and intermediate lecturer of BGI's Animal and Plant Molecular Breeding Department. In 2014, he joined Shanghai Lingen Biotechnology as the head of the bioinformatics department, responsible for the management and training of bioinformatics personnel, the handling of after-sales problems of projects, and the development and optimization of analysis processes. Interested teachers and students are welcome to communicate and cooperate with us.