Scientia Agricultura Sinica ›› 2026, Vol. 59 ›› Issue (3): 556-574.doi: 10.3864/j.issn.0578-1752.2026.03.007

• PLANT PROTECTION • Previous Articles     Next Articles

Construction of an RNAi-Enhanced Metarhizium anisopliae Targeting PxGNBP3 and Its Immunoregulatory Mechanism in Plutella xylostella

YAN WenYing1(), ZHANG YuanZhen1(), WU HongXin1, PANG Rui1, CHEN ZePeng2, JIN FengLiang1(), XU XiaoXia1()   

  1. 1 College of Plant Protection, South China Agricultural University/State Key Laboratory of Green Pesticide, Guangzhou 510642
    2 China National Tobacco Corporation Guangdong Provincial Branch, Guangzhou 510610
  • Received:2025-10-13 Accepted:2025-11-18 Online:2026-02-01 Published:2026-01-31
  • Contact: JIN FengLiang, XU XiaoXia

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Abstract:

【Objective】Gram-negative binding proteins (GNBPs), also known as β-1,3-glucan recognition proteins, represent a class of crucial pattern recognition receptors (PRRs) in insects and play pivotal roles in the innate immune response. This study aimed to systematically identify members of the PxGNBP gene family in the diamondback moth (Plutella xylostella), analyze their structural characteristics and expression patterns, as well as screen and validate key target genes. The findings are expected to reveal the innate immune mechanisms and evolutionary adaptations of P. xylostella in response to pathogenic infection, thereby providing theoretical foundations and potential targets for the development of novel biological control. 【Method】Based on the whole-genome data of P. xylostella, members of the PxGNBP gene family were identified. Bioinformatic approaches were comprehensively employed to analyze their structural characteristics and evolutionary relationships, and AlphaFold3 was used to predict their three-dimensional structures. In addition, combined with public transcriptome data and quantitative real-time polymerase chain reaction (RT-qPCR) technology, the expression patterns of these family members in different tissues and post-infection with Beauveria bassiana and Metarhizium anisopliae were detected. Recombinant M. anisopliae strains carrying pSilent-PxGNBP3 were constructed. The expression levels of PxGNBP3 and downstream antimicrobial peptide genes post-infection were determined via RT-qPCR, and the pathogenicity of different strains against P. xylostella was evaluated using bioassays. 【Result】A total of 10 PxGNBP members were identified in P. xylostella. Among them, PxβGRP4 is located on chromosome 22 and belongs to the glucanase subfamily, while the remaining 9 members are located on chromosome 29 and belong to the PRR subfamily. Phylogenetic and chromosome location analyses suggested the occurrence of tandem duplication events within this gene family. Conserved motif analysis indicated that the N-terminal domain of PxGNBP exhibited lower conservation compared to the C-terminal domain. Except for PxβGRP4, the key catalytic sites of glucanase in other members were mutated. Three-dimensional structure predictions revealed that all members, except PxβGRP4 and PxβGRP3, possessed the typical GNBP protein structure; the C-terminus of PxβGRP3 contained a structural fragment that was similar but not identical to Carbohydrate-binding module 39 (CBM39). Expression profile analysis demonstrated that most members exhibited a time-series expression pattern of first increasing and then decreasing after infection with the two fungi. RNA interference (RNAi) assays showed that the recombinant M. anisopliae strains could effectively suppress the expression of PxGNBP3, leading to a significant reduction in antimicrobial peptide expression levels and a decrease in host survival rate. Moreover, the virulence of recombinant strains was significantly higher than that of the wild-type strain and enhanced with increasing concentration. 【Conclusion】Ten members of the GNBP gene family were identified in P. xylostella, with PxβGRP3 and PxGNBP3-2 showing structural specificity. This gene family exhibited a time-series regulatory expression pattern in response to fungal infection. In vivo functional validation of PxGNBP3 via RNAi was successfully achieved using the constructed recombinant M. anisopliae strains. The results provide important insights for elucidating the innate immune mechanisms of P. xylostella and developing novel targets for biological control.

Key words: Gram-negative binding protein (GNBP), Plutella xylostella, Metarhizium anisopliae, RNAi, biological control

Table 1

The primer sequences used in this study"

基因Gene 引物及序列Primer and sequence (5′-3′) 基因Gene 引物及序列Primer and sequence (5′-3′)
PxβGRP4 PxβGRP4_F: CGTTATGGCAGCAAGATG PxGNBP3 PxGNBP3-Xho I F1: GCG CTCGAGCGGAACACAAGATTGCTTTG
PxβGRP4_R: TTACGACCGTTCCAGAAG PxGNBP3-Hind III R1: GCC AAGCTTTCAAGTTGCCTCTGGCTGT
PxβGRP3 PxβGRP3_F: GTATACGGCAACGGTCGCTA PxGNBP3-Kpn I F2: GCC GGTACCCGGAACACAAGATTGCTTTG
PxβGRP3_R: AATCGTTCGATCCGCCTACC PxGNBP3-Sph I R2: GCC GCATGCTCAAGTTGCCTCTGGCTGT
PxGNBP3-1 PxGNBP3-1_R: CAGCAGCCAATAGCGGTTTC GFP GFP-Xho I F1: CCG CTCGAGATGGTGAGCAAGGGCGAG
PxGNBP3-1_F: TGGCTACAAACCAGGGCATT GFP-Hind III R1: AGC AAGCTTTACTTGTACAGCTCGTCCATGCC
PxGNBP3-2 PxGNBP3-2_R: AACTCCTTGTTCCACTCTTC GFP-Kpn I F2: CGG GGTACCATGGTGAGCAAGGGCGAG
PxGNBP3-2_F: GACGGTAACTACGCATCC GFP-Sph I R2: ACAT GCATGCTACTTGTACAGCTCGTCCATGCC
PxGNBP3-3 PxGNBP3-3_R: ATCATTGGACCCACCGACAC Moricin Mor-qF: GTCAACGTCAACGCCCTCAAG
PxGNBP3-3_F: GACCTCAGGGACACAACTGG Mor-qR: GTCCACCCCTGGCACTGTCTA
PxGNBP3 PxGNBP3_R: ATACTATGAGGTCTGGTGTC Lysozyme1 Lys1-qF: CGCATTGTCTGAAGGGAAGGT
PxGNBP3_F: CAAGATTACGGCAATAAGGT Lys1-qR: TTGATCTGGAACAGCCCGTAG
PxGNBP3-5 PxGNBP3-5_R: TGTTCTCGGTGTCATTCG Lysozyme2 Lys2-qF: CCCTGAAAGTAGTTGCTGGAT
PxGNBP3-5_F: CGCAGATTGAGGCACTAT Lys2-qR: GCCTCTACACGGCTTCGTTAT
PxGNBP3-6 PxGNBP3-6_R: CCGTTGGTTGTTGTTCCT Cecropin1 Cep1-qF: TATACATTATTTAACCCGTAAAT
PxGNBP3-6_F: CTGGTCATCTCCGTATGTG Cep1-qR: GTCGCTGTCATCGGACAAGCCAC
PxGNBP2 PxGNBP2_R: GCATGTGTTGGTTAAGTCC Cecropin2 Cep2-qF: CATCACGGATGTGCTGTCCCACT
PxGNBP2_F: TCCTGAAGAGCCTGATTATC Cep2-qR: GGAATAAAAGATTCCAATTTCAA
PxβGRP1 PxβGRP1_R: CGACGAATCCTTCAACAGT Cecropin3 Cep3-qF: TACTTCTTCTTCACGGTTGTCG
PxβGRP1_F: TCCAGAGACATCACCAGAC Cep3-qR: CCCAATATGCTGGATGCTTGTC
RPS13 RPS13-F: TCAGGCTTATTCTCGTCG Gloverin Glo-qF: TGTGCCGTGGCTCAAGTTTC
RPS13-R: GCTGTGCTGGATTCGTAC Glo-qR: CCTGACGGTAGCCCGCCTTA

Fig. 1

Phylogenetic tree of GNBP proteins from P. xylostella and other insects"

Fig. 2

Chromosome localization (A) and collinearity analysis (B) of GNBP gene family in P. xylostella"

Fig. 3

Conserved motif of P. xylostella GNBP family genes"

Fig. 4

Multiple sequence alignment of P. xylostella GNBP family genes Those highlighted with a red background are the key structural sites of the glucanase GH16 domain"

Fig. 5

Protein three-dimensional structures of GNBP family gene members from P. xylostella The region previously identified as the GH16 domain；红色Red：前期鉴定为信号肽的区域The region previously identified as the signal peptide"

Fig. 6

Temporal expression of GNBP family genes in P. xylostella of different tissues and following B. bassiana infection"

Fig. 7

Expression patterns of GNBP family genes in P. xylostella in response to M. anisopliae infection"

Fig. 8

The construction of recombinant strains of M. anisopliae based on RNAi"

Fig. 9

Expression levels of antimicrobial peptide genes in P. xylostella after infection with different Metarhizium strains"

Fig. 10

Toxicity of different M. anisopliae strains to P. xylostella"

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