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Plant Commun
Plant Commun
Plant Communications
2590-3462
Elsevier

S2590-3462(24)00282-7
10.1016/j.xplc.2024.100974
100974
Research Article
A faster killing effect of plastid-mediated RNA interference on a leaf beetle through induced dysbiosis of the gut bacteria
Zhang Yiqiu 1
Ke Zebin 1
Xu Letian 1
Yang Yang 1
Chang Ling lingchang@hubu.edu.cn
1∗
Zhang Jiang zhangjiang@hubu.edu.cn
12∗∗
1 State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Hongshan Laboratory, School of Life Sciences, Hubei University, Wuhan 430062, China
2 Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Key Laboratory of Synthetic Biology, Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518000, China
∗ Corresponding author lingchang@hubu.edu.cn
∗∗ Corresponding author zhangjiang@hubu.edu.cn
14 5 2024
09 9 2024
14 5 2024
5 9 10097427 12 2023
10 4 2024
10 5 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The expression of double-stranded RNAs (dsRNAs) from the plastid genome has been proven to be an effective method for controlling herbivorous pests by targeting essential insect genes. However, there are limitations to the efficiency of plastid-mediated RNA interference (PM–RNAi) due to the initial damage caused by the insects and their slow response to RNA interference. In this study, we developed transplastomic poplar plants that express dsRNAs targeting the β-Actin (dsACT) and Srp54k (dsSRP54K) genes of Plagiodera versicolora. Feeding experiments showed that transplastomic poplar plants can cause significantly higher mortality in P. versicolora larvae compared with nuclear transgenic or wild-type poplar plants. The efficient killing effect of PM–RNAi on P. versicolora larvae was found to be dependent on the presence of gut bacteria. Importantly, foliar application of a gut bacterial strain, Pseudomonas putida, will induce dysbiosis in the gut bacteria of P. versicolora larvae, leading to a significant acceleration in the speed of killing by PM–RNAi. Overall, our findings suggest that interfering with gut bacteria could be a promising strategy to enhance the effectiveness of PM–RNAi for insect pest control, offering a novel and effective approach for crop protection based on RNAi technology.

This study reports that the willow leaf beetle can be effectively controlled by expressing dsRNA from the poplar plastid genome to target two essential insect genes. The insect-killing effect of plastid-mediated RNAi can be further enhanced by foliar application of a specific gut bacterial strain. The potential mechanism involves alteration and overgrowth of the gut bacteria and reduced immune responses of the insects.

Key words

poplar
RNA interference
gut bacteria
plastid transformation
Plagiodera versicolora
Published: May 14, 2024
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pmcIntroduction

RNA interference (RNAi) is a post-transcriptional regulation mechanism triggered by double-stranded RNAs (dsRNAs) and is highly conserved in most eukaryotic organisms (Fire et al., 1998; Zhu and Palli, 2019). RNAi technology has proven to be a valuable tool in reverse genetics for studying gene function. It has also shown great potential in insect pest management (Mohr et al., 2010; Kim et al., 2015). When dsRNAs targeted against essential insect genes are delivered into insect cells, the endogenous RNAi machinery is activated. This leads to stunted growth, impaired fecundity, and even death of the insect. Although nuclear transgenic plants expressing dsRNA targeted against essential insect genes have been successful in increasing resistance to target insects, efficient protection of the plants and efficient killing of the insects have not been achieved, likely owing to the low expression and instability of dsRNAs caused by plants’ own Dicer cleavage (Mao et al., 2007; Paim et al., 2013; Zhang et al., 2017; Cai et al., 2018; Hussain et al., 2019). Recently, a breakthrough was achieved through plastid-mediated RNA interference (PM–RNAi) technology. This approach involves the expression of long insecticidal dsRNAs in plastids, which lack an RNAi machinery and can accumulate dsRNAs at a high level. This strategy has been effective in combating pests such as Colorado potato beetle (Leptinotarsa decemlineata), 28-spotted potato ladybird (Henosepilachna vigintioctopunctata), western flower thrips (Frankliniella occidentalis), and spider mites (Zhang et al., 2015; Wu et al., 2022a, 2022b; Xu et al., 2022). However, even with the use of PM–RNAi, there is still considerable damage caused by pests during the initial feeding period. This is likely due to the slow response of the target pests to RNAi. Therefore, further improvements are needed to enhance the efficiency of PM–RNAi.

Poplar trees are highly valued as renewable and sustainable resources for fuel, fiber, and wood. They are known for their fast growth and wide distribution (Stobrawa, 2014), making them an ideal plant for studying forest tree physiology and genetic improvement (Ye et al., 2011; Polle et al., 2013). However, these trees often face severe damage and even death caused by insect pests. Significant progress has been made in poplar genetic engineering breeding to cultivate insect-resistant varieties. This involves introduction of genes from Bacillus thuringiensis (Bt) into the nuclear and plastid genomes of poplar (McCown et al., 1991; Wu et al., 2019). Such genetic modification has proven effective in combating pests and protecting the health of poplar plantations. Among the many insect pests that pose a threat to poplar trees, Plagiodera versicolora is particularly noteworthy. This forest pest primarily feeds on the leaves of Salicaceae plants and is widely distributed across Asia, Europe, and northern Africa (Urban, 2005; Yoneya et al., 2014). Both the larvae and adults of P. versicolora feed on the mesophyll of the leaves, causing them to curl and turn brown and potentially leading to the death of the affected trees.

The insect gut microbiota not only play vital roles in the coevolution of symbiotic interactions mediated by secondary metabolites with their hosts (Liberti and Engel, 2020; Zhang et al., 2024) but also engage in complex interactions such as symbiosis, commensalism, and pathogenicity (Xiong, 2022; Zhang et al., 2023). Throughout different stages of insect development, the gut serves as a dynamic environment for microorganisms. When the multispecies community of microorganisms is destroyed, the microbes are potentially harmful to the host and provide a new microbial control against insects (Mason et al., 2011; Wei et al., 2017; Xu et al., 2019; Sun et al., 2023). Furthermore, research has demonstrated that certain insect gut microbiota can enhance the insecticidal activity of Bt toxin (Broderick et al., 2009; Caccia et al., 2016; Lei et al., 2023). Previously, we discovered that ingestion of dsRNA by insects caused a dysbiosis of the gut bacterial community. Remarkably, the degradation products of dsRNA were utilized by the gut bacteria for their growth, consequently contributing to the potent insecticidal activity of dsRNA against P. versicolora larvae (Xu et al., 2021). On the basis of these findings, we hypothesized that leveraging the insect gut bacteria could enhance the efficacy of PM–RNAi against P. versicolora.

In this study, we initially generated transgenic poplar plants that express dsRNAs targeting P. versicolora β-Actin and Srp54k genes (Zhang et al., 2019). Our results demonstrated that transplastomic poplar plants can cause significantly higher mortality in P. versicolora larvae compared with nuclear transgenic and wild-type poplar plants. Interestingly, the presence of gut bacteria in P. versicolora larvae is required for the heightened efficacy of PM–RNAi, and foliar application of P. versicolora gut bacteria can significantly improve the insect resistance of poplar to P. versicolora through PM–RNAi.

Results

Generation and molecular characterization of transplastomic and nuclear transgenic poplar plants

Based on our previous RNAi target-gene screening (Zhang et al., 2019), we selected the β-Actin and Srp54k genes of P. versicolora as the two essential targets for construction of plastid transformation vectors to express dsRNAs in poplar plastids and nuclear transformation vectors to express hairpin RNAs (hpRNAs) from the poplar nuclear genome, respectively. β-Actin encodes the essential cytoskeletal protein β-actin (Zhang et al., 2015). Srp54k encodes signal recognition particle protein 54k, a key component of the ribonucleoprotein complex that mediates the co-translational targeting of secretory and membrane proteins to the endoplasmic reticulum (Liu et al., 2002; Ulrich et al., 2015). dsACT and dsSRP54K, which target the P. versicolora β-Actin and Srp54k genes, were produced by transcription of two convergent Prrn promoters from tobacco (Figure 1A). The two plastid constructs were introduced into poplar plastids by biolistic bombardment (Wu et al., 2019; Xu et al., 2020), and the two nuclear constructs were introduced into the poplar nuclear genome by Agrobacterium-mediated transformation (Wang et al., 2011). We obtained two independent transplastomic poplar lines for each plastid transformation construct (Pt-pt-dsACT#1, Pt-pt-dsACT#2, Pt-pt-dsSRP54K#1, and Pt-pt-dsSRP54K#2), which were used for further analysis. Integration of the transgenes into the plastid genome was confirmed by Southern blot analyses. All transformed lines showed the expected hybridization signals resulting from transgene integration into the plastid genome by homologous recombination (Pt-pt-dsACT, 4.29 kb; Pt-pt-dsSRP54K, 4.26 kb) and the complete absence of the hybridization signal at 3.5 kb that was diagnostic of the wild-type plastid DNA (Figure 1B, 1C and Supplemental Figure 1A). The expression of dsACT and dsSRP54K in the transplastomic poplar plants was confirmed by northern blot analyses. Through comparison with a dilution series of in vitro-synthesized RNA, the levels of dsACT and dsSRP54K were estimated to be approximately 0.42% and 0.19% of total cellular RNA, respectively (Figure 1D and 1E).Figure 1 Generation and molecular characterization of transplastomic and nuclear transgenic poplar plants.

(A) Physical maps of transformation vectors for dsRNA expression from the plastid genome of poplar. The cassettes designed to produce dsRNAs are schematically depicted below the map, along with the expected structures and sizes. The selectable marker gene aadA is driven by the psbA promoter combined with the 5′ untranslated region (5′ UTR) from gene 10 of bacteriophage T7 (CrPpsbA:T7g10) and the 3′ UTR of the rbcL gene from Chlamydomonas reinhardtii (CrTrbcL). The location of the hybridization probe is shown as a black bar.

(B and C) Southern blot analysis of transplastomic poplar lines Pt-pt-dsACT(B) and Pt-pt-dsSRP54K(C). DNA samples were digested with BglII and hybridized to a Digoxin (DIG)-labeled probe as indicated. Absence of the 3.50-kb hybridization signal for the wild-type genome indicates homoplasmy of transplastomic lines.

(D and E) Quantification of dsACT(D) and dsSRP54K(E) accumulation in transplastomic poplar plants by northern blot analysis. Five micrograms of total cellular RNA were loaded from transplastomic and wild-type poplar plants in each lane. For semi-quantitative analysis, a dilution series of in vitro-synthesized dsRNAs was loaded for comparison. The GelView-stained gel prior to blotting is shown below the blot as a loading control.

(F and G) The relative expression of hpACT(F) and hpSRP54K(G) in the nuclear transgenic poplar lines was determined by qRT–PCR. The poplar EF1β gene was used as an internal standard. Data are shown as means ± SEM (n = 3).

From eight independent nuclear-transformed lines for each nuclear transformation construct, we selected two lines that exhibited high expression levels (Pt-nu-hpACT#18, Pt-nu-hpACT#36, Pt-nu-hpSRP54K#7, and Pt-nu-hpSRP54K#24) for subsequent experiments (Figure 1F, 1G, and Supplemental Figure 1B). Although transplastomic poplar plants accumulated large amounts of dsRNAs, no hpRNAs were detected in nuclear transgenic poplar plants (Supplemental Figure 2A and 2B). Moreover, small interfering RNAs (siRNAs) were detectable only in nuclear transgenic poplar plants but not in transplastomic poplar plants (Supplemental Figure 2C and 2D). All transplastomic and nuclear transgenic poplar plants grew normally and displayed wild-type-like phenotypes, as indicated by their height and chlorophyll content (Supplemental Figure 3; t-test, p > 0.05).

Assessment of the resistance of transplastomic and nuclear transgenic poplar plants to P. versicolora larvae

To assess the levels of insect resistance in transplastomic and nuclear transgenic poplar plants, we performed bioassays using first-instar P. versicolora larvae. We monitored the survival rates, mean weights, and leaf area consumed for insects that fed on detached leaves of transplastomic, nuclear transgenic, and wild-type poplar (Pt-wt) plants. The transplastomic poplar plants had significantly higher insect mortality than the wild-type and nuclear transgenic poplar plants. All larvae feeding on Pt-pt-dsACT lines were dead after 7 days (Figure 2A), whereas those feeding on Pt-pt-dsSRP54K lines were dead after 8 days (Supplemental Figure 4A). By contrast, none of nuclear transgenic poplar plants caused larval mortality. Transplastomic poplar plants also resulted in significantly reduced larval growth compared with nuclear transgenic poplar plants (Figure 2B, ANOVA, p < 0.05; Supplemental Figure 4B, ANOVA, p < 0.05). Similarly, the damage and leaf consumption caused by P. versicolora larvae on transplastomic poplar plants was minimal at 3 days for Pt-pt-dsACT lines (Figure 2C and 2D) and 4 days for Pt-nu-hpSRP54K lines (Supplemental Figure 4C and 4D), whereas the damage to nuclear transgenic poplar leaves was comparable to that of wild-type poplar leaves. As expected, feeding on transplastomic poplar plants significantly suppressed the expression of β-Actin (Figure 2E) and Srp54k (Supplemental Figure 4E) genes in P. versicolora larvae at 3 and 4 days, respectively. By contrast, feeding on nuclear transgenic lines had no significant effect on β-Actin at 3 days (Figure 2E, ANOVA, p > 0.05), although there was a reduction in gene expression at later time points (day 4) (Figure 2F, ANOVA, p < 0.05). By contrast, larvae that fed on leaves of nuclear transgenic lines expressing hpSRP54K showed no significant suppression of Srp54k expression at 4 days (Supplemental Figure 4E, ANOVA, p > 0.05) or 5 days (Supplemental Figure 4F, ANOVA, p > 0.05).Figure 2 Feeding assays of first-instar P. versicolora larvae on transplastomic and nuclear transgenic poplar lines expressing dsACT.

(A) Kaplan–Meier survival curves of first-instar larvae (n = 30) fed on detached leaves of Pt-wt, nuclear transgenic (Pt-nu-hpACT#18 and Pt-nu-hpACT#36), and transplastomic (Pt-pt-dsACT#1 and Pt-pt-dsACT#2) poplar plants. The log-rank test was used to assess the significance of differences between two survival curves. The statistical significance (p < 0.05) is denoted by different letters. The p values for each pair of plant lines are shown.

(B) Mean weight of larvae after 3 days of feeding on detached poplar leaves.

(C) Leaves damaged by larvae after 3 days of feeding. Data are means ± SE (three biological replicates; 10 insects per sample). The letters above the bars indicate the statistical significance of differences between the groups (p < 0.05, ANOVA with Bonferroni multiple-comparison test). The damaged area was determined with Image J.

(D) Representative example of bioassays with detached leaves of the indicated poplar plants. Leaves were exposed to first-instar larvae and replaced with fresh leaves every day; the photograph was taken at day 3. The diameter of the culture dish was 9 cm. Relative expression levels of β-Actin in larvae fed with the indicated poplar leaves at 3 (E) and 4 (F) days were determined by qRT–PCR. Gene expression levels in larvae fed on detached leaves of Pt-wt were set to 1. Data are means ± SE (three biological replicates; one insect per sample). Different letters above the bars indicate significant differences between groups (p < 0.05, ANOVA with Bonferroni multiple-comparison test).

When directly comparing the insect resistance of transplastomic poplar plants expressing dsACT and dsSRP54K, we observed that larvae fed on leaves of Pt-pt-dsACT were killed significantly faster than those fed on leaves of Pt-pt-dsSRP54K (Supplemental Figure 5A, log-rank test, p < 0.05). This finding was further supported by the reduced leaf damage (Supplemental Figure 5B–5D) caused by P. versicolora larvae on detached leaves of Pt-pt-dsACT compared with those of Pt-pt-dsSRP54K. These results suggest that transplastomic poplar plants expressing dsRNA targeting the β-Actin gene had superior protection against P. versicolora compared with those targeting the Srp54k gene and/or the nuclear transgenic poplar plants.

Gut bacteria are required for efficient PM–RNAi in P. versicolora larvae

We previously demonstrated that gut bacteria play a role in accelerating dsRNA-induced mortality in P. versicolora larvae (Xu et al., 2021). To investigate whether gut bacteria also influence the efficiency of PM–RNAi, we compared non-axenic and axenic larvae fed with detached aseptic leaves of Pt-pt-dsACT and Pt-wt. Non-axenic larvae fed with the aseptic leaves of Pt-pt-dsACT died significantly faster than axenic larvae (Figure 3A, log-rank test, p < 0.0001), but there were no significant differences in survival between axenic and non-axenic larvae fed with leaves of Pt-wt (Figure 3A, log-rank test, p = 0.691). The level of β-Actin mRNA was notably reduced in larvae after 2 days (Figure 3B, t-test, Pt-wt [axenic] vs. Pt-pt-dsACT [axenic], p < 0.05; Pt-wt [non-axenic] vs. Pt-pt-dsACT [non-axenic], p < 0.05) and 3 days (Figure 3B, t-test, Pt-wt [axenic] vs. Pt-pt-dsACT [axenic], p < 0.05; Pt-wt [non-axenic] vs. Pt-pt-dsACT [non-axenic], p < 0.05) of feeding on aseptic leaves of Pt-pt-dsACT, but the extent of gene silencing did not differ significantly between axenic and non-axenic larvae after 2 days (Figure 3B, t-test, p = 0.856) or 3 days (Figure 3B, t-test, p = 0.061). This suggests that the striking differences in mortality between non-axenic and axenic larvae fed with leaves of Pt-pt-dsACT were not due to differences in suppression of the target gene. Overall, our findings indicate that the presence of gut bacteria is involved in accelerating PM–RNAi in non-axenic P. versicolora larvae.Figure 3 Gut bacteria accelerate the mortality of P. versicolora larvae in PM–RNAi.

(A) Kaplan–Meier survival curves of axenic and non-axenic first-instar larvae (n = 30) after feeding on detached aseptic poplar leaves of Pt-wt and Pt-pt-dsACT plants. The log-rank test was used to assess the significance of differences between two survival curves. ∗∗∗p < 0.001; ∗p < 0.05; NS, not significant.

(B) Relative expression levels of β-Actin in axenic and non-axenic larvae fed with detached aseptic leaves of Pt-wt and Pt-pt-dsACT plants after 2 and 3 days, as determined by qRT–PCR. Data are mean ± SE (three biological replicates; one insect per sample). Different letters indicate significant differences as determined by t-test (p < 0.05).

(C) Kaplan–Meier survival curves of axenic first-instar larvae (n = 30) fed with detached aseptic leaves of Pt-wt and Pt-pt-dsACT plants after reintroduction of P. putida by painting onto aseptic poplar leaves at a concentration of about 1 × 104 CFU/cm2. The log-rank test was used to assess the significance of differences between two survival curves. ∗∗∗p < 0.001; ∗p < 0.05; NS, not significant.

(D) Relative expression levels of β-Actin in axenic larvae fed with detached aseptic poplar leaves of Pt-wt and Pt-pt-dsACT plants after 2 and 3 days of reintroduction of P. putida, as determined by qRT–PCR. Data are means ± SE (three biological replicates; one insect per sample). Different letters indicate significant differences as determined by t-test (p < 0.05).

To investigate the effect of gut bacteria on the resistance of transplastomic poplar plants to P. versicolora larvae, we painted P. putida, a gut bacterial species previously found in the gut of P. versicolora larvae, onto detached leaves of Pt-pt-dsACT. These leaves were then fed to first-instar axenic larvae. We observed that the mortality rate of axenic larvae fed on the P. putida-inoculated leaves of Pt-pt-dsACT was significantly higher than that of axenic larvae (Figure 3C, log-rank test, p < 0.0001). However, we did not observe any significant differences in gene silencing levels between the axenic larvae and the P. putida-inoculated larvae after 2 days (Figure 3D, t-test, p = 0.400) or 3 days (Figure 3D, t-test, p = 0.660). These findings suggest that reintroduction of P. putida into axenic P. versicolora larvae can enable their effective control by PM–RNAi, similar to that observed for non-axenic larvae.

Foliar application of P. putida enhances the resistance of transplastomic poplar plants to P. versicolora

Because P. versicolora is a non-axenic species in nature, we wanted to investigate whether gut bacteria, specifically P. putida, could be used as a reinforcing agent to improve the efficiency of PM–RNAi against non-axenic P. versicolora. To this end, we fed first-instar non-axenic larvae with leaves of Pt-pt-dsACT coated with a series of concentrations of P. putida (10, 102, 103, 104, and 105 colony-forming units [CFU]/cm2). Foliar application of P. putida led to faster killing of insects compared with the control group (foliar application of H2O; Supplemental Figure 6, log-rank test, p < 0.05). When non-axenic larvae were fed with detached leaves of Pt-pt-dsACT coated with P. putida at a concentration of 102 CFU/cm2 (a dose known to accelerate mortality), they were killed significantly faster than larvae fed on detached leaves of Pt-pt-dsACT coated with H2O (Figure 4A, log-rank test, p < 0.0001). Similarly, non-axenic larvae fed with detached leaves of Pt-pt-dsSRP54K coated with P. putida were also killed significantly faster than axenic larvae (Supplemental Figure 7A, log-rank test, p < 0.05). Moreover, the foliar application of P. putida resulted in less leaf damage after feeding compared with the control group (Figure 4B and 4C; Supplemental Figure 7B and 7C). However, there were no significant differences in gene suppression levels between the control non-axenic larvae and P. putida-introduced non-axenic larvae after 2 days (Figure 4D, t-test, Pt-pt-dsACT vs. Pt-pt-dsACT+P. putida: p = 0.400) or 3 days (Figure 4D, t-test, Pt-pt-dsACT vs. Pt-pt-dsACT+P. putida: p = 0.660) when fed with leaves of Pt-pt-dsACT or after 3 days (Supplemental Figure 7D, t-test, Pt-pt-dsSRP54K vs. Pt-pt-dsSRP54K + P. putida: p = 0.806) or 4 days when fed with leaves of Pt-pt-dsSRP54K (Supplemental Figure 7D, t-test, Pt-pt-dsSRP54K vs. Pt-pt-dsSRP54K + P. putida: p = 0.990). The striking differences in mortality between P. putida-introduced and control non-axenic larvae fed with transplastomic poplar expressing lethal dsRNAs (dsACT or dsSRP54K) are therefore not caused by differences in suppression of the target genes. These findings suggest that P. putida can be used to enhance the efficiency of PM–RNAi against P. versicolora.Figure 4 Foliar application of gut bacteria enhances the resistance of transplastomic poplar plants to P. versicolora larvae.

(A) Kaplan–Meier survival curves of first-instar non-axenic larvae (n = 30) fed with detached aseptic poplar leaves of Pt-wt and Pt-pt-dsACT plants coated with P. putida. The poplar leaves were painted with P. putida at a concentration of about 1 × 102 CFU/cm2. The log-rank test was used to assess the significance of differences between two survival curves. ∗∗∗p < 0.001; NS, not significant.

(B) Representative example of bioassays with detached leaves of the indicated poplar plants. Leaves were exposed to first-instar larvae and replaced with fresh leaves every day; the photographs were taken separately at 2 and 3 days. The diameter of the culture dish was 9 cm.

(C) Leaves damaged by larvae after 2 and 3 days of feeding. Data are means ± SE (three biological replicates; 10 insects per sample). The letters above the bars indicate the significance of differences as determined by t-test (p < 0.05). The damaged area was determined with ImageJ.

(D) Relative expression levels of ACT in larvae fed with indicated poplar leaves at 2 and 3 days, as determined by qRT–PCR. Gene expression levels in larvae fed on detached leaves of Pt-wt were set to 1. Data are means ± SE (three biological replicates; one insect per sample). Different letters above the bars indicate significant differences, as determined by t-test (p < 0.05).

Alteration and overgrowth of the gut bacteria in P. versicolora contribute to the enhanced efficiency of PM–RNAi

Because P. putida is a commensal in the healthy guts of P. versicolora larvae, insect survival (Supplemental Figure 8A, log-rank test, p = 0.930) and body mass (Supplemental Figure 8B, t-test, p = 0.157) did not differ significantly between P. putida-introduced and control non-axenic larvae. Western blot analysis revealed no difference in actin levels between P. putida-introduced and control non-axenic larvae fed with leaves of Pt-pt-dsACT after 2 days (Supplemental Figure 9). Histological analysis also showed disruption of the peritrophic matrix and gut epithelium in both P. putida-introduced and control non-axenic larvae fed with leaves of Pt-pt-dsACT. However, there were no differences in intestinal integrity between P. putida-introduced and control non-axenic larvae fed with leaves of Pt-wt or Pt-pt-dsACT (Supplemental Figure 10).

To examine whether breakdown of the gut epithelium barrier is responsible for the increased efficiency of dsRNA-mediated killing, first-instar non-axenic larvae were fed with detached leaves of Pt-wt that had been painted with a mixture of dsACT and P. putida. These larvae were killed significantly faster, with all larvae dying within 5 days, than larvae fed on detached leaves of Pt-wt coated with dsACT alone (Supplemental Figure 11, log-rank test, p < 0.05). There was no effect on the mortality of larvae fed with detached leaves of Pt-wt coated with dsGFP, which served as the control (Supplemental Figure 11, log-rank test, p = 0.485).

The insect hemocoel, a body cavity filled with blood, is home to a large number of hemocytes (blood cells), as well as a diverse array of soluble immune effectors. Once some gut bacteria make their way from the gut lumen into the hemocoel, they may shift from harmless symbionts to harmful pathogens. This triggers a strong immune response in the insect, leading to rapid death of the larvae (Mason et al., 2011). Immunofluorescence analysis showed that the gut epithelia of non-axenic larvae fed with leaves of Pt-pt-dsACT coated with P. putida:GFP were disrupted, and fluorescence signals were detected in the hemocoel (Supplemental Figure 12). This indicates the translocation of gut bacteria from the gut epithelium into the hemocoel in P. versicolora larvae fed with leaves of Pt-pt-dsACT.

We next investigated the composition of the gut bacterial community in P. versicolora and how it changed when P. putida was introduced to transplastomic poplar plants. Sterilized water was used as a negative control. We characterized the amplified bacterial 16S rRNA sequences using next-generation sequencing. Alpha diversity was assessed using four metrics: Chao1, Observed_otus, Shannon, and Simpsons index. No significant differences were observed in the Chao1 and Observed_otus index when comparing gut samples from the four groups (Supplemental Table 2, t-test, p > 0.05). However, the Shannon index of the Pt-pt-dsACT and Pt-pt-dsACT + P. putida groups were notably lower than those of the Pt-wt and Pt-wt + P. putida groups (Supplemental Table 2, t-test, p < 0.05). No significant difference was observed between the Pt-pt-dsACT and Pt-pt-dsACT + P. putida groups. Similarly, the Simpson index showed significantly lower diversity in the Pt-pt-dsACT and Pt-pt-dsACT + P. putida groups compared with the Pt-wt and Pt-wt + P. putida groups (Supplemental Table 2, t-test, p < 0.05). However, there was no significant difference in Simpson index between the Pt-pt-dsACT and Pt-pt-dsACT + P. putida groups. These results suggest that feeding on Pt-pt-dsACT plants contributes to a reduction in diversity of the associated microbiota of P. versicolora larvae. Principal coordinates analysis (PCoA) of Bray–Curtis distances of the microbial communities revealed that the bacterial communities of the four groups clustered separately (Figure 5A). Nonmetric multidimensional scaling (NMDS) based on Bray–Curtis distances also produced a similar pattern (Figure 5B). As shown in the Venn diagram, 548, 811, 415, and 320 unique genera were detected in the Pt-wt, Pt-wt + P. putida, Pt-pt-dsACT, and Pt-pt-dsACT + P. putida groups, respectively, and 64 genera were consistently found in all gut samples (Figure 5C). These findings clearly indicate a decrease in diversity and significant alterations in community structure of the gut microbial population in larvae feeding on Pt-pt-dsACT plants. In larvae fed with leaves of Pt-wt, the dominant bacterial genera in the gut community were Lactobacillus, Enterobacter, Pseudomonas, and Serratia. Remarkably, compared with those of the control group (fed with leaves of Pt-wt), the composition and diversity of the gut bacterial population changed markedly in non-axenic larvae fed with leaves of Pt-pt-dsACT. Specifically, when larvae were fed with leaves of Pt-pt-dsACT, there was a notable increase in the proportion of Enterobacter and a significant decrease in the proportion of Lactobacillus relative to those of larvae fed with leaves of Pt-wt, as shown by the relative abundances of amplicon sequence variants (ASVs) (Figure 5D, t-test, p < 0.05).Figure 5 Combined use of PM–RNAi and gut bacteria results in alteration of the gut bacteria and promotes their growth in P. versicolora.

(A) Principal coordinate analysis (PCoA) based on the Bray–Curtis metric of 20 samples (five biological replicates; 10 guts per sample) for the bacterial community.

(B) Nonmetric multidimensional scaling (NMDS) based on Bray–Curtis distances.

(C) A Venn diagram was used to illustrate the shared and unique genera among the different treatment groups.

(D) Relative abundances of the top 10 genera in the four indicated groups of gut bacteria in P. versicolora larvae. The relative abundances of taxa that could not be annotated to the genus level have been excluded from these plots.

(E–H) The absolute abundance of total bacteria (E), Pseudomonas(F), Enterobacter(G), and Lactobacillus(H) in P. versicolora larvae fed with leaves of the indicated plants, as determined by qRT–PCR. The measurements were performed separately at 24 and 48 h after feeding. The qRT–PCR values for larvae fed on detached leaves of Pt-wt at 24 and 48 h were set to 1. The unit of the vertical axis is copies/sample. Data are means ± SE (three biological replicates; 10 larval guts per sample). Different letters above the bars indicate significant differences, as determined by t-test (p < 0.05).

The gut bacterial load of P. versicolora larvae was analyzed using qRT–PCR. The total amount of gut bacteria was significantly higher in P. putida-introduced non-axenic larvae fed with leaves of Pt-pt-dsACT than in the other three groups after 24 and 48 h (Figure 5E, t-test, p < 0.05). These observations suggest that the introduction of P. putida greatly promotes the growth of the gut bacteria in non-axenic larvae fed with leaves of Pt-pt-dsACT. To further confirm the effects of P. putida introduction on the absolute abundance of the gut bacterial community, the absolute quantities of bacterial genera were examined in different groups of larvae. The absolute abundance of Pseudomonas was significantly higher in P. putida-introduced non-axenic larvae fed with the leaves of Pt-wt or Pt-pt-dsACT than in control non-axenic larvae at 24 h, and Pseudomonas levels were significantly lower in non-axenic larvae fed with the leaves of Pt-pt-dsACT, either with or without P. putida introduction, at 48 h (Figure 5F, t-test, p < 0.05). The absolute abundance of Enterobacter was significantly higher at 48 h in both P. putida-introduced and control non-axenic larvae fed with leaves of Pt-pt-dsACT than in control non-axenic larvae fed with leaves of Pt-wt (Figure 5G, t-test, p < 0.05). When larvae were fed with leaves of Pt-pt-dsACT, there was an increase in the absolute abundance of Enterobacter between P. putida-introduced non-axenic and control non-axenic larvae (Figure 5G). Although the difference did not reach statistical significance according to the t-test (p = 0.074), there was a noticeable trend toward higher abundance of Enterobacter in the P. putida-introduced non-axenic larvae fed with leaves of Pt-pt-dsACT. No significant differences were found in the absolute abundance of Lactobacillus between the P. putida-introduced and control non-axenic larvae fed with leaves of Pt-pt-dsACT at 24 or 48 h (Figure 5H, t-test, p > 0.05). These findings indicate that the introduction of P. putida leads to changes in the gut bacteria and increases the gut bacterial load in P. versicolora larvae, enhancing the efficiency of PM–RNAi against P. versicolora.

Introduction of P. putida enhances the resistance of transplastomic poplar plants to P. versicolora by reducing its immune responses

The immune defense response is rapidly activated when intestinal epithelial cells are under attack by pathogens. Furthermore, an imbalance in gut homeostasis can also trigger an immune response, including the immune deficiency (IMD), Toll, Janus kinase signal transducer and activator of transcription (JAK/STAT), and dual oxidase (Duox)–reactive oxygen species (ROS) regulatory pathways (Zeng et al., 2022). Antimicrobial peptides (AMPs) are generated mainly through the IMD and Toll pathways, and studies indicate that AMPs in the insect gut originate primarily from the IMD signaling pathway (Neyen et al., 2012). The JAK/STAT signaling pathway also participates in intestinal immune regulation by influencing the production of AMPs (Hoffmann, 2003). In addition, AMPs and ROS have antibacterial activity, producing ROS, with the activation of Duox leading to the generation of ROS that can directly destroy ROS-sensitive microorganisms (Ha et al., 2005).

We next measured the expression of AMP-encoding (Attacin and Defensin) and ROS-related (Duox) genes of P. versicolora by qRT–PCR analysis. Compared with control non-axenic larvae fed with leaves of Pt-wt, P. putida-introduced non-axenic larvae fed with leaves of Pt-pt-dsACT showed no significant changes in Attacin expression at 48 h (Figure 6A, t-test, p = 0.316). However, Attacin was significantly upregulated at 48 h in control non-axenic larvae fed with leaves of Pt-pt-dsACT compared with P. putida-introduced non-axenic larvae fed with leaves of Pt-pt-dsACT (Figure 6A, t-test, p < 0.05). Defensin expression increased at 24 and 48 h in both P. putida-introduced and control non-axenic larvae fed with leaves of Pt-pt-dsACT compared with control non-axenic larvae fed with leaves of Pt-wt (Figure 6B, t-test, p < 0.05). Similarly, Duox expression was significantly upregulated in both P. putida-introduced and control non-axenic larvae fed with leaves of Pt-pt-dsACT at 24 and 48 h. There was also a significant difference between P. putida-introduced and control non-axenic larvae fed with leaves of Pt-pt-dsACT at 48 h (Figure 6C, t-test, p < 0.05). The reduced Duox expression was consistent with the weaker ROS signal in the gut of P. putida-introduced non-axenic larvae fed with leaves of Pt-pt-dsACT at 48 h (Supplemental Figure 13, t-test, p < 0.05). In summary, these findings suggest that introduction of P. putida reduces the production of ROS and the expression of AMP-encoding genes, limiting their ability to constrain the overgrowth of gut bacteria. This mechanism could ultimately enhance the resistance of transplastomic poplar plants to P. versicolora.Figure 6 Gene expression patterns of immune-related genes in P. versicolora larvae after feeding on the indicated plants for 24 or 48 h, as measured by qRT–PCR.

The mRNA expression levels of the AMP-encoding genes Attacin(A) and Defensin(B); the ROS-related gene Duox(C); the effector-encoding gene Lysozyme(D); the recognition-factor-encoding gene PGRP(E); and the signal-transduction factor-encoding genes Relish(F), Spätzle(G), and Domeless(H) were examined. The mRNA expression levels in larvae fed on detached leaves of Pt-wt at 24 or 48 h were set to 1. Data are means ± SE (three biological replicates; 10 larval guts per sample). Different letters above the bar indicate significant differences, as determined by t-test (p < 0.05).

The expression of Lysozyme, an insect effector-encoding gene, was significantly downregulated at 24 h and upregulated at 48 h in larvae that were fed with leaves of Pt-pt-dsACT and were either introduced with P. putida or left untreated. Interestingly, the expression of Lysozyme was lower in P. putida-introduced larvae than in control non-axenic larvae fed with leaves of Pt-pt-dsACT (Figure 6D, t-test, p < 0.05). Similarly, the gene encoding peptidoglycan recognition protein (PGRP) was significantly upregulated in both P. putida-introduced and untreated larvae fed with leaves of Pt-pt-dsACT at 48 h (Figure 6E, t-test, p < 0.05).

Genes encoding signal transduction factors (Relish, Domeless, Spätzle) were also significantly upregulated in both P. putida-introduced and untreated larvae fed with leaves of Pt-pt-dsACT at 48 h (Figure 6F–6H, t-test, p < 0.05). Notably, the expression of Relish and Domeless was lower in P. putida-introduced larvae than in untreated larvae fed with leaves of Pt-pt-dsACT at 48 h (Figure 6F–6H, t-test, p < 0.05). This result suggests that transcription of AMP-encoding genes may be downregulated by the introduction of P. putida through the IMD and JAK/STAT pathways (Buchon et al., 2009; Yu et al., 2022), thus enhancing the resistance of transplastomic poplar plants to P. versicolora.

Discussion

In this study, we aimed to develop transplastomic poplar plants that were highly resistant to P. versicolora by expression of dsACT or dsSRP54K in plastids. We found that plastid-expressed dsRNA accumulated to high levels, as plastids lack an RNAi pathway (Zhang et al., 2015). However, the resistance of nuclear transgenic plants against insects was found to be very low (Supplemental Figure 2 and Supplemental Figure 4), likely because of the low expression of dsRNAs and the instability of dsRNAs in the nucleus. dsRNAs that are expressed from the nuclear genome can be processed into siRNAs by Dicer-like protein, but these siRNAs are much less effective than dsRNAs in inducing an RNAi response in coleopteran insects (Zhang et al., 2017).

We observed that the accumulation of dsACT in young leaves of Pt-pt-dsACT lines reached approximately 0.42% of total cellular RNA. This high dsACT accumulation resulted in high toxicity to P. versicolora larvae (Figures 1D and 2). On the other hand, the low hpACT accumulation in Pt-nu-hpACT lines failed to induce strong RNAi effects (Figure 2 and Supplemental Figure 2). These findings demonstrate that transplastomic poplar plants expressing dsRNAs can greatly enhance the mortality of P. versicolora and can be used to enhance the effectiveness of RNAi-based pest control. These results also suggest that conventional nuclear transformation technology is not as effective as PM–RNAi, likely owing to the low expression of dsRNAs and the instability of dsRNAs in the nucleus.

It has been observed that coleopteran insects are more susceptible to RNAi than insects of other orders (Huvenne and Smagghe, 2010) because of factors such as cellular uptake in the gut environment and dsRNA stability in the digestive system, which is affected by enzymatic degradation and high pH (Cooper et al., 2019). Our data indicate that P. versicolora is highly susceptible to RNAi, and Pt-pt-dsACT lines showed higher resistance against first-instar P. versicolora larvae (which died within 6 days) than did Pt-pt-dsRP54K lines (Supplemental Figure 5). Previous studies have found that targeting the highly conserved β-Actin gene by PM–RNAi is effective in causing high mortality in L. decemlineata and H. vigintioctopunctata, making it a potent target (Zhang et al., 2015; Xu et al., 2022). However, the insecticidal effects of PM–RNAi in transplastomic potato plants were reduced in older larvae or adults (Zhang et al., 2015).

It has been reported that there is a crosstalk between the gut microbiota and insecticidal dsRNAs in P. versicolora, suggesting a potential role for gut bacteria in RNAi efficiency (Xu et al., 2021). The degradation products of dsRNA can be used by gut bacteria for their growth, contributing to dsRNA-induced insecticidal activity. In this work, we used plant tissue culture to obtain aseptic poplar leaves for rearing axenic larvae from surface-sterilized eggs, with non-axenic larvae as a control (Ma et al., 2021). Our results indicated that non-axenic larvae were killed significantly faster than axenic larvae when feeding on aseptic leaves of Pt-pt-dsACT (Figure 3A). This effect was independent of the efficiency of gene knockdown, ruling out the possibility that differences in mortality were caused by different levels of target-gene silencing (Figure 3B). Furthermore, reintroduction of P. putida (which had the strongest effect on accelerating larval mortality) into axenic larvae increased their mortality after feeding on leaves of Pt-pt-dsACT (Figure 3C and 3D).

Many bacteria isolated from insects belong to the families Bacillaceae, Enterobacteriaceae, and Pseudomonaceae. These bacterial insect pathogens have been used successfully in insect control (Thiery and Frachon, 1997; Sezen et al., 2007; Ferreira et al., 2019). The Pseudomonas genus, in particular, is known for its plant-growth-promoting abilities and its potential for use in agriculture (Beneduzi et al., 2012; Bhattacharyya and Jha, 2012). P. putida has been found to be nitrogen fixing and capable of promoting plant growth. It has also shown insecticidal activity, with a strain isolated from L. decemlineata being 100% effective against larvae within 5 days (Muratoğlu et al., 2014; Sun et al., 2022). P. putida is a gram-negative, rod-shaped bacterium that is often found in soil, water, and plants, especially in contaminated environments (Weimer et al., 2020). Because of its rapid growth, robustness upon challenge with oxidative stress and toxins, and ability to be easily manipulated for genetic studies, P. putida is considered to be a valuable subject for research on soil bacteria, bacteria-mediated soil processes, and environmental applications (Timmis, 2002; Poblete-Castro et al., 2012). When fed with leaves of either Pt-pt-dsACT or Pt-pt-dsSRP54K, first-instar P. putida-introduced larvae died significantly faster than untreated larvae (Figure 4 and Supplemental Figure 7). These results suggest that P. putida can accelerate the mortality of P. versicolora larvae fed with transplastomic poplar plants expressing lethal dsRNA. Furthermore, we observed significant changes in the composition and overgrowth of gut bacteria when larvae were fed with leaves of Pt-pt-dsACT, with or without P. putida (Figure 5). These results suggest that the faster killing effect of PM–RNAi on P. versicolora may be due to induced dysbiosis of the gut bacteria. It is important to note that although P. putida is a commensal bacterium in the gastrointestinal tracts of healthy animals and does not cause harm in that environment, its translocation from the gut epithelia to the hemocoel can lead to sepsis and rapid death in P. versicolora larvae (Xu et al., 2021). In line with previous findings, we also observed translocation of P. putida:GFP from the gut epithelia to the hemocoel in larvae fed with leaves of Pt-pt-dsACT coated with P. putida:GFP (Supplemental Figure 12). This result further supports the idea that P. putida plays a role in the observed dysbiosis and rapid killing of P. versicolora larvae.

Diversity of the gut bacterial community decreased in larvae fed with leaves of Pt-pt-dsACT (Supplemental Table 2; Figure 5C). In addition, the relative abundance of Enterobacter, which was the dominant bacterial genus, was notably higher in larvae fed with leaves of Pt-pt-dsACT (Figure 5D). This increase in Enterobacter abundance was a consequence of the disruption of the larvae’s gut epithelium caused by feeding on leaves of Pt-pt-dsACT. As a result, the opportunistic bacterial pathogen Enterobacter outgrew other bacteria in these larvae in comparison to control non-axenic larvae fed with leaves of Pt-wt. The gut bacterial load also increased significantly in P. versicolora larvae fed with leaves of Pt-pt-dsACT coated with or without P. putida (Figure 5E). Moreover, the absolute abundance of Enterobacter was higher in P. putida-introduced non-axenic larvae than in control non-axenic larvae fed with leaves of Pt-pt-dsACT (Figure 5G). These findings suggest that Enterobacter may have stronger and more negative effects on insect performance when the peritrophic matrix and gut epithelium are disrupted, ultimately leading to the rapid death of P. versicolora larvae.

The innate immune system of insects is usually responsible for defending against pathogenic microorganisms through the release of ROS and the expression of AMPs. However, when P. versicolora larvae were fed with leaves of Pt-pt-dsACT, the bacterial load in their intestines increased significantly over time, especially in P. putida-introduced non-axenic larvae (Figure 5D). The gut immune barrier, a chitinous multilayered structure, is the first line of defense against pathogenic microorganisms. It induces a local response in insects to prevent invasion and reduce mortality (Kuraishi et al., 2011). Our results revealed that the introduction of P. putida can strongly stimulate the immune system of P. versicolora larvae (Figure 6). Lysozyme also play a crucial role in the immune defense against bacteria and fungi (Mittapalli et al., 2006). The expression of Lysozyme was significantly lower in P. putida-introduced larvae than in untreated larvae fed with leaves of Pt-pt-dsACT after 48 h (Figure 6D). We also found that the expression of Relish and Domeless was significantly lower in P. putida-introduced larvae than in untreated larvae fed with leaves of Pt-pt-dsACT after 48 h (Figure 6F and 6H). These results suggest that introduction of P. putida can downregulate the transcription of AMP-encoding genes through the IMD and JAK/STAT pathways. The rapid death of P. versicolora larvae may be a result of immune suppression, which can lead to dysbiosis of the gut bacteria and an increase in their virulence.

On the basis of our findings, we have proposed a schematic model for the introduction of P. putida to enhance PM–RNAi-mediated resistance by inducing dysbiosis of the gut bacteria in P. versicolora (Figure 7). Our study demonstrates that PM–RNAi is an effective strategy for controlling pests in forestry and is superior to dsRNA expression from the nuclear genome. We found that high levels of dsRNAs accumulate in the plastids of transplastomic poplar plants and are enclosed in the chloroplasts, in contrast to nuclear transgenic poplar plants in which dsRNAs are processed into siRNAs by Dicer-like protein. Oral delivery of large quantities of dsRNAs leads to reduced levels of target mRNA and mortality in P. versicolora. Furthermore, transcription of AMP-encoding and ROS-related genes was downregulated by introduction of P. putida into non-axenic larvae, resulting in an immune deficiency. Specifically, transcription of AMP-encoding genes is downregulated by Relish and Domeless, which may limit the immune responses of the larvae and hinder their ability to control the overgrowth of gut bacteria. In this model, both Relish and Domeless may serve as transcription factors involved in the IMD and JAK/STAT pathways, which regulate insect tolerance to gut bacteria.Figure 7 A schematic model illustrating the introduction of gut bacteria to enhance the resistance of PM–RNAi in  P. versicolora.

The ingestion of long dsRNAs expressed in plastids leads to a reduction in target mRNA levels and is highly toxic to P. versicolora larvae. In comparison to control non-axenic larvae fed with leaves of Pt-pt-dsACT, P. putida-introduced non-axenic larvae exhibit lower production of ROS and reduced transcription of AMP-encoding genes, leading to immunodeficiency (IMD). Moreover, the expression of AMPs is regulated by transcription of Relish and Domeless through IMD and JAK/STAT pathways, in turn suppressing immune responses. This limited immune response hampers the ability of P. versicolora to control the overgrowth of gut bacteria.

Numerous studies have provided evidence that Relish, an important member of the nuclear factor κB transcription factor family, is a master regulator of AMP-encoding gene expression in insects, including Drosophila, in response to pathogenic infections (Ertürk-Hasdemir et al., 2009; Shi et al., 2015). Relish mRNA level was highest in the gut tissue of Tenebrio molitor larvae infected with the gram-negative bacterium Escherichia coli, and knockdown of Relish significantly increased mortality (Keshavarz et al., 2020). Here, our data demonstrated that Relish expression was induced in response to gut bacterial infection in a manner similar to that observed in T. molitor (Figure 6F). In addition to Relish, the JAK/STAT pathway has also been identified as an important player in the immune response of Drosophila to pathogenic infections. This pathway is responsible for synthesizing antibacterial and antifungal peptides such as Drosomycin and in promoting epithelial renewal through cell proliferation to maintain gut homeostasis in the face of infection (Khan et al., 2023). The JAK/STAT pathway has three pivotal components: the cytokine-like transmembrane receptor Domeless, the JAK kinase Hopscotch, and the transcription factor STAT92E. Activation of this pathway occurs through binding of unpaired ligands to Domeless, leading to the aggregation of JAK, phosphorylation, and nuclear translocation of STAT for regulation of AMP expression (Zeng et al., 2022). Our results revealed that expression of Domeless was downregulated in P. putida-introduced larvae compared with control larvae fed with leaves of Pt-pt-dsACT at 48 h (Figure 6H). It is possible that an excess of ROS caused by intestinal damage may have activated JAK/STAT pathways in control non-axenic larvae fed with leaves of Pt-pt-dsACT at 48 h, leading to initiation of intestinal repair and immune defense responses (Figure 6C and 6H).

In summary, we successfully used PM–RNAi technology to control forestry pests and developed a novel strategy to enhance the effects of RNAi-based pest control by utilizing the gut bacteria of the target pest. These findings not only highlight the potential of P. putida as a reinforcement agent to improve the effectiveness of RNAi against insect pathogens but also shed light on the mechanisms underlying this enhanced killing effect through dysbiosis and translocation of gut bacteria.

Methods

Insect rearing

P. versicolora larvae and adults were collected from a field at Sha Lake Park in Wuhan (30.35°N, 114.33°E), Hubei Province, China. The insects were fed with fresh leaves of willow (Salix babylonica L.) in an insectary at 28°C, 60% ± 5% relative humidity, and a photoperiod of 14-h light/10-h dark.

Vector construction

The poplar plastid transformation vectors were constructed from the previously described vectors pYY25 and pJZ237 (Zhang et al., 2015; Wu et al., 2019). To begin, poplar genomic DNA was used as a template to amplify the right homologous recombination region (RHRR), which contains the psbZ and trnG genes. The RHRR was amplified using the R-F/R-R primer pair and was subsequently digested with SacI and NheI. Next, the left homologous recombination region (LHRR), which consists of portions of psaB, rps14, and trnfM genes, was PCR amplified with the primer pair L-F/L-R. The PCR product was digested with SalI and KpnI. The DNA fragment containing the aadA expression cassette was digested with SpeI and SalI from the plasmid pYY25 (Wu et al., 2019). The backbone plasmid pBluescript II KS (+) was digested with SacI and KpnI. A ligation reaction involving the four DNA fragments (RHRR, LHRR, the aadA expression cassette, plasmid backbone) was performed with T4 DNA ligase. The resulting ligation products were transformed into E. coli competent cells (XL10-Gold) to generate the plasmid pYY19. To eliminate the SacI site, the plasmid pYY19 was digested with SacI, blunted with T4 DNA polymerase, and self-ligated with T4 DNA ligase, generating the plasmid pYQ20. The ACT expression cassette was excised from pJZ237 by digestion with SalI and XbaI. It was then cloned into the plasmid pYQ20 digested with SalI and SpeI, generating the plasmid pYY118. ACT (350 bp) and SRP54K (340 bp) fragments originating from the β-Actin and Srp54k genes of P. versicolora larvae were obtained by PCR amplification with the primer pairs ACT-F(SbfI)/ACT-R(SacI) and SRP54K-F(SbfI)/SRP54K-R(SacI) using the bacterial expression vectors pL4440-ACT and pL4440-SRP54K as templates, respectively (Zhang et al., 2019). The ACT fragments of L. decemlineata were excised from pYY118 by digestion with SbfI and SacI and were replaced by PCR products, which were digested with SbfI and SacI, resulting in the plastid transformation vectors pRNAi-dsACT and pRNAi-dsSRP54K. The psbA promoter of the aadA gene cassette (CrPpsbA) (digested with BglII and ApaI) was replaced by the psbA promoter fused to the 5′ untranslated region (UTR) of gene 10 from bacteriophage T7 (CrPpsbA: T7g10), generating the plastid transformation vector pRNAi-dsACT-T7g10.

The poplar nuclear transformation vectors were constructed from the previously described vector pHW25 (Fu et al., 2021). The first ACT-f (350 bp) and SRP54K-f (340 bp) fragments were amplified with the primer pairs ACT-350f-F/ACT-350f-R and SRP54K-340f-F/SRP54K-340f-R (Supplementary Table 1), introducing PstI and NheI restriction sites. The GA20-intron sequence was amplified with the primer pair GA20-intron-F/GA20-intron-R from pUC-RNAi, introducing EcoRI and SpeI restriction sites. The second ACT-r (350 bp) and SRP54K-r (340 bp) fragments, in the antisense orientation, were amplified with primer pairs ACT-350r-F/ACT-350r-R and SRP54K-340r-F/SRP54K-340r-R (Supplementary Table 1), introducing EcoRI and NotI restriction sites. The PCR products were digested with the corresponding restriction enzymes and cloned into the similarly cut plasmid pHW25, yielding the poplar nuclear transformation vectors pRNAi-hpACT and pRNAi-hpSRP54K. The primers used for PCR are listed in Supplementary Table 1.

Poplar plastid and nuclear transformation

Poplar plastid transformation was performed as described previously. In brief, fresh young leaves of 4-week-old poplar were selected and bombarded with plasmid DNA-coated gold particles (0.6 μm) using a PDS-1000/He Delivery System and 900-psi rupture discs (Bio-Rad, Hercules, CA, USA). After bombardment, the leaf explants were cut into 3 × 3-mm pieces. Primary spectinomycin-resistant events were selected on the PaSIM1 medium containing 30 mg l−1 spectinomycin, and then transferred onto PaSIM2 medium containing 30 mg l−1 spectinomycin for shoot elongation (Wu et al., 2019). Primary events were verified by PCR and Southern blotting analysis. The independent transplastomic lines were subjected to several rounds of regeneration on spectinomycin-containing PaSIM2 for selection of homoplasmy. Poplar nuclear transformation was performed by Agrobacterium-mediated transformation according to a previous report (Wang et al., 2011). Nuclear transgenic poplar lines were identified by antibiotic selection and confirmed by qRT–PCR. Elongation factor 1-beta (EF1β) was used as the internal reference gene for determining gene expression in poplar (Yang et al., 2015). The growth conditions for the whole selection procedure were 16-h light (20–25 μE m−2 s−1) at 25°C and 8-h dark at 20°C in a growth chamber.

RNA isolation and qRT–PCR

Total RNA was isolated from plants and insects using RNAiso Plus Reagent (Takara, Dalian, China) following the manufacturer’s instructions. Samples (1 μg) of total RNA were used for first-strand cDNA synthesis with Hifair II 1st Strand cDNA Synthesis Kit (Yeasen, Shanghai, China). The qPCR reaction was performed using a CFX Connect Real-Time System (Bio-Rad, Hercules, CA, USA) with SYBR Premix Ex Taq II (Tli RNaseH Plus, Takara, Dalian, China). The reaction mixture for PCR amplification consisted of 2 μl of cDNA, 5 μl of 2 × SYBRfi Premix Ex Taq II, 0.25 μl of forward and reverse primers (10 μM), and 2.5 μl of sterilized ultrapure H2O in a final 10-μl reaction volume. The qPCR program included an initial denaturation step at 95°C for 2 min, followed by 40 cycles at 95°C for 5 s, 60°C for 30 s, and 72°C for 30 s. Ribosomal protein S18 (RPS18) was selected as the reference gene for normalizing gene expression, and the relative expression levels were determined using the 2−ΔΔCt method (Zhang et al., 2019).

Samples of P. putida-introduced or control non-axenic larvae (containing gut bacteria) fed with leaves of Pt-wt or Pt-pt-dsACT were collected at 24 and 48 h. The larvae were disinfected in 70% ethanol for 10 s, rinsed in sterile water three times, and then dissected under sterile conditions. Ten guts of larvae were pooled as a replicate (n = 3), and their total RNA was extracted for absolute quantification of gut bacteria. Standard curves were generated using serial dilutions of plasmid DNA containing target fragments (Schwarz et al., 2016; Lim et al., 2018). The primers used for the quantification of gut bacteria and the expression of immune-related genes are listed in Supplementary Table 1. All experiments were repeated with three independent biological sample preparations.

Isolation of nucleic acids and gel blot analysis

Total cellular DNA was isolated from fresh leaves using a cetyltrimethylammonium bromide (CTAB)-based method. For Southern blot analysis, 5 μg of total cellular DNA was digested with BglII (Takara, Dalian, China). The digested DNA was separated by 1% agarose gel electrophoresis and transferred onto a positively charged nylon membrane (GE Healthcare, Chicago, IL USA). To verify plastid transformation, a 587-bp fragment of the psaB gene was PCR amplified from poplar plastid genomic DNA with the primer pair Pt-Probe-psaB-F/Pt-Probe-psaB-F. Labeling of the probe and hybridization were performed with the DIG-High Prime DNA Labeling and Detection Starter Kit II following the manufacturer’s instructions (Roche, Basel, Switzerland).

For RNA gel blot analysis, 5 μg of total RNA was denatured and then separated by electrophoresis in formaldehyde-containing 1% agarose gels. After separation, the RNA was transferred onto Hybond nylon membranes. Probes used to detect the accumulation of dsACT and dsSRP54K in transplastomic poplar plants were PCR amplified using pRNAi-ACT-T7g10 or pRNAi-SRP54K as templates with the primer pairs T7-N-ACT-F/N-ACT-R and T7-N-SRP54K-F/N-SRP54K-R, respectively. To examine the dsRNA expression levels in transplastomic poplar plants, young leaves with a leaf plastochron index (Erickson and Michelini, 1957; Taylor et al., 2003) of 0.89 ± 1, 0.79 ± 1, 0.93 ± 1, 0.5 ± 1, and 0.72 ± 1 for Pt-wt, Pt-pt-dsACT#1, Pt-pt-dsACT#2, Pt-pt-dsSRP54K#1, and Pt-pt-dsSRP54K#2, respectively, were sampled for measurement of dsRNA accumulation by northern blot analysis.

For siRNA analysis, 60-μg samples of total cellular RNA were separated in 14% polyacrylamide gels with 0.3 M sodium acetate and 7 M urea as the gel buffer and 0.3 M sodium acetate (pH 5.0) as the running buffer. The separated RNA samples were electroblotted onto Hybond nylon membranes in a blotting buffer containing 10 mM Tris–acetate (pH 7.8), 5 mM sodium acetate, and 0.5 mM embryonic testis differentiation homolog A. The electroblotting was performed at 20 V for 2 h at 4°C, and the RNA was cross-linked to the membrane by UV light. PCR products produced using the primer pairs siN-Probe-ACT-F/siN-T7-Probe-ACT-R and siN-T7-Probe-SRP54K-F/siN-Probe-SRP54K-R (Supplementary Table 1) were used as probes to detect siACT or siSRP54K accumulation in poplar. The probes were labeled with DIG using the DIG-High Prime DNA Labeling and Detection Starter Kit I, following the manufacturer’s instructions (Roche, Basel, Switzerland).

Insecticidal activity of transgenic poplar plants

Synchronized first-instar larvae (n = 30) were selected and divided into three groups. Detached leaves of wild-type and transgenic poplar plants were used for bioassays. Larval mortality was recorded daily, and leaves consumed by insects were photographed. The consumed leaves were replaced with fresh leaves every day. Another group of insects was treated in the same way and subjected to RNA extraction. All experiments were repeated three times.

Reintroduction of gut bacteria into axenic (gut-bacteria-free) P. versicolora

Axenic larvae were obtained as described previously with minor modifications (Xu et al., 2021). In brief, newly laid eggs were carefully collected from willow leaves with a brush pre-dipped in 0.75% NaOH, followed by soaking in 75% ethanol for 7 min. They were then washed with sterilized water three times. After air-drying, each egg was individually transferred onto Luria–Bertani (LB) solid medium. The newly hatched axenic larvae were fed with aseptic poplar leaves. To explore the influence of gut bacteria removal on P. versicolora, first-instar axenic and non-axenic larvae were fed with detached aseptic leaves of wild-type and transplastomic poplar plants, separately. Insect mortality was monitored daily. Another group of insects underwent the same treatment and were sampled for RNA extraction.

To reintroduce gut bacteria into axenic P. versicolora, P. putida (a previously isolated gut bacterial species from P. versicolora, accession number MT791337) was cultured in LB medium at 28°C for 12–24 h, then centrifuged at 6000 g for 5 min to remove residual LB medium (Xu et al., 2021). The bacterial pellets were washed twice with sterilized PBS, resuspended in PBS, and diluted to a concentration of approximately 1 × 106 CFU/ml by counting CFUs on LB medium. First-instar axenic P. versicolora larvae were fed with aseptic detached leaves of poplar plants that had been coated with P. putida at a concentration of 104 CFU/cm2. Sterilized PBS-painted leaves were used as the control.

Effect of exogenously applied gut bacteria on non-axenic P. versicolora

To determine the optimal concentration of P. putida for enhancing the efficiency of PM–RNAi against P. versicolora, we tested a series of P. putida concentrations (10, 102, 103, 104, 105 CFU/cm2) by applying bacterial suspensions onto fresh poplar leaves. The leaves were then fed to first-instar non-axenic larvae. Mortality was recorded daily, and leaves consumed by insects were photographed.

To assess the efficiency of PM–RNAi against P. versicolora, first-instar non-axenic larvae (n = 30) were fed with detached poplar leaves that had been painted with P. putida (102 CFU/cm2). The leaves were replaced daily, and the survival rate was recorded. To assess the efficacy of RNAi-mediated knockdown of target genes, an additional group of identically fed larvae was raised, three larvae were randomly selected each day after dsRNA feeding, and total RNA was isolated for further analysis.

DNA fragments of the β-Actin and gfp genes were PCR amplified using gene-specific primer pairs (Supplementary Table 1) and used as templates for synthesis of dsRNA (dsACT and dsGFP) using the T7 RiboMAX Express RNAi system (Promega, USA) following the manufacturer’s instructions. dsRNA integrity was evaluated by electrophoresis in 1% agarose gels, and dsRNA amounts were quantified with a spectrophotometer (NanoDrop 2000, Thermo Scientific, USA).

Protein extraction and western blotting

Total proteins from P. putida-introduced and control non-axenic larvae fed with leaves of Pt-wt or Pt-pt-dsACT were isolated following an established protocol (Cahoon et al., 1992). The protein concentration was determined using a BCA protein assay reagent kit (TransGen Biotech, China). Samples (10 μg) of total proteins were separated by 10% SDS–PAGE using the Omni-Easy One Step PAGE Gel Fast Preparation Kit (EpiZyme, China), and the gels were either stained with Coomassie blue for direct visualization or transferred to a polyvinylidene difluoride (PVDF) membrane (GE Healthcare, Chicago, IL USA). After blocking with 5% nonfat dry milk, the membrane was incubated with a 3000-fold diluted polyclonal antibody against β-actin (ABclonal, China) for 90 min and then incubated for 90 min with a 5000-fold diluted horseradish peroxidase (HRP)-conjugated secondary antibody (Bioswamp, China). Immunobiochemical detection was performed using the ECL chemiluminescence reagent kit (Biosharp, China) according to the manufacturer’s instructions.

Histological analysis

Histological analysis was carried out as described previously (Xu et al., 2021). In brief, whole bodies of P. versicolora larvae were collected and immediately fixed in a formalin–acetic acid–alcohol liquid solution for 24 h. The fixed samples were dehydrated in an ethanol series (beginning with 50% and progressing to 100%), incubated sequentially in an ethanol/xylene mixture (1:1; v/v) and 100% xylene, cleared in xylol for 4 h, and finally embedded in paraffin. To observe the gut epithelia of larvae and assess the efficiency of RNAi-mediated knockdown of target genes in P. versicolora, paraffin sections (approximately 3 μm thick) were prepared from the larval samples and stained with TRITC-Phalloidin (Yeasen, Shanghai, China). The sections were analyzed under a fluorescence microscope (Nikon Eclipse E-200, Japan).

To investigate whether breakdown of the barrier function of the gut epithelium is responsible for the increased killing efficiency, we transformed the P. putida strain with the pBBR1B403-GFP plasmid stably expressing GFP (P. putida:GFP) (Xiao et al., 2016). After feeding larvae with transplastomic poplar leaves coated with P. putida:GFP, we collected the larvae for immunofluorescence analysis after 48 h. Cross sections were permeabilized with 0.1% Triton X-100 for 10 min, blocked with 1% BSA for 30 min, and labeled with anti-eGFP antibody (Servicebio, Wuhan, China) overnight at 4°C. Alexa Fluor 488 goat anti-rabbit IgG/FITC (Servicebio, Wuhan, China) was used at a 1:400 dilution in PBS for 50 min at room temperature to detect GFP fluorescence (green). β-Actin (red) was stained with TRITC-Phalloidin (Servicebio, Wuhan, China). The images were analyzed with a fluorescence microscope (Nikon Eclipse C2, Tokyo, Japan) with excitation wavelengths of 545 and 488 nm and emission wavelengths of 570 and 511 nm, respectively.

Analysis of gut bacteria

Samples of second-instar larvae were collected 48 h after feeding on detached leaves of wild-type and transplastomic poplar plants with or without P. putida. The larvae were disinfected in 70% ethanol for 10 s, rinsed in sterile water three times, and dissected under sterile conditions. Ten guts were pooled together as one replicate (n = 5) and cryopreserved at −80°C for subsequent experiments. Midgut bacterial genomic DNA was extracted from gut samples using the Blood/Cell/Tissue DNA kit (Biomarker, China) following the manufacturer’s instructions.

Bacterial communities from all samples were analyzed by sequencing the hypervariable region of the 16S rRNA gene (V3 and V4 region) on the Illumina NovaSeq platform. The raw sequences were demultiplexed according to their unique barcodes, and adaptor and primer sequences were removed using QIIME (Caporaso et al., 2010). The paired-end reads were merged using FLASH (Version 1.2.11, http://ccb.jhu.edu/software/FLASH/). The taxonomic assignment of ASVs was performed using the VSEARCH consensus taxonomy classifier in QIIME 2 with reference datasets from the SILVA 16S rRNA database (Callahan et al., 2016). Any unclassified ASVs were manually assigned to taxonomic groups at the genus level using the NCBI database. ASVs with an abundance of <5 reads were removed. Microbial diversity and community composition were analyzed using vegan packages in R (version 3.5.3) (Li et al., 2020). NMDS and PCoA based on Bray–Curtis distances were used to identify differences between microbial communities. Compositional differences in NMDS were tested using analysis of similarities with 1000 permutations. The top 10 species with the highest abundance at the genus level were selected for each group, and a column sum plot of relative abundance was generated to visually examine the species with high relative abundance and their proportions at different taxonomic levels of each group. The sequencing data have been deposited at the NCBI Sequence Read Archive under BioProject PRJNA1048681.

In vivo detection of ROS

After 48 h of feeding with leaves of Pt-wt or Pt-pt-dsACT, the guts of P. putida-introduced and control non-axenic larvae were dissected in PBS containing 10 μM dihydroethidium (Servicebio, Wuhan, China). After incubation in the dark for 30 min at room temperature, the guts were washed twice with PBS buffer and immediately examined under a fluorescence microscope (Nikon Eclipse C2, Tokyo, Japan) with excitation wavelengths of 520 and 340 nm and emission wavelengths of 605 and 488 nm, respectively.

Statistical analysis

Survival curves were analyzed using the Kaplan–Meier method, and the log-rank test was used to evaluate the significance of differences between two groups. Data comprising two groups were analyzed using Student’s t-test for unpaired comparisons, and data comprising more than two groups were analyzed using one-way ANOVA with the Bonferroni (equal variances) or Dunnett’s T3 (unequal variances) procedure for multiple comparisons. A value of p < 0.05 was considered statistically significant. Data were statistically analyzed with SPSS version 19.0. Figures were drawn with GraphPad Prism 6. Damaged areas were determined with Image J. Figures were assembled in Adobe Illustrator CS5 and Adobe Photoshop CS6.

Funding

This work was supported by grants from the 10.13039/501100001809 National Natural Science Foundation of China (32271912 , 32272634 , 32101484 ).

Author Contributions

J.Z. designed the research. Y.Z., Z.K., and Y.Y. performed the research. Y.Z., L.X., Y.Y., L.C., and J.Z. analyzed the data. L.C. and J.Z. wrote the paper with input from the other authors.

Supplemental information

Document S1. Supplemental Figures 1‒13 and Supplemental Tables 1 and 2

Document S2. Article plus supplemental information

Acknowledgments

No conflict of interest is declared.

Published by the Plant Communications Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and CEMPS, CAS.

Supplemental information is available at Plant Communications Online.
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