ABOUT US
Empowering Science with Precision and Simplicity!
Literature Sharing│FaNDUFB9 Attenuates Strawberry Mottle Virus Infection by Inhibiting the Activity of the Viral Gene Silencing Suppressor, Pro2Glu
Release time:
2025-03-17
Strawberry mottle virus (SMoV) significantly compromises strawberry fruit quality and yield. The Pro2Glu protein of SMoV, an RNA silencing suppressor, is pivotal for viral infection; however, its interactions with host factors remain underexplored. This study identifies a critical interaction between SMoV Pro2Glu and strawberry NADH dehydrogenase (ubiquinone) 1 β subcomplex subunit 9 (FaNDUFB9). Using yeast two-hybrid assays and in vivo protein interaction analyses, we confirm that amino acid residues 31–45 of FaNDUFB9 are essential for binding Pro2Glu. Sequence analysis places FaNDUFB9 within the Complex1_LYR_superfamily, and subcellular localisation reveals its presence in both the mitochondria and nucleus. Silencing of NbNDUFB9 in Nicotiana benthamiana elevated Pro2Glu expression, whereas co-expression of FaNDUFB9 with Pro2Glu suppressed Pro2Glu levels and weakened its silencing activity. Furthermore, overexpression of FaNDUFB9 in transgenic N. benthamiana reduced transient SMoV RNA expression. These findings suggest that FaNDUFB9 inhibits expression of Pro2Glu, affecting its silencing activity, and thereby potentially further influencing viral infection.
The Pro2Glu protein encoded by SMoV functions as a silencing suppressor. To elucidate its mechanism during SMoV infection, yeast two-hybrid (Y2H) screening was performed using Pro2Glu as bait. Initial analyses confirmed that Pro2Glu lacks self-activation activity (Figure S1). A cDNA library was screened, referencing the transcripts from Fragaria × ananassa ‘Camarosa’ Genome Assembly (v1.0.a1), which identified 12 positive signals (Table S1). Subsequent validation of these candidates through Y2H assays confirmed the interactions of FaERDJ3 (maker-Fvb7-1-snap-gene-320.152-meRNA-1) and FaNDUFB9 (snap_masked-Fvb2-2-processed gen-157.21-mRNA-1), with FaNDUFB9 selected for further study based on functional analyses (Figure S2).
Using artificial intelligence modelling provided by Pronetbio (Nanjing, China), the three-dimensional structures of Pro2Glu and FaNDUFB9 were simulated, resulting in a high-confidence interaction score of 0.9395 (Figure 1a), indicating a strong likelihood of binding between the two proteins.
FIGURE 1 | Identification of the interaction between Pro2Glu and FaNDUFB9
FaNDUFB9, a subunit of mitochondrial complex I, was identified as a potential interaction partner of Pro2Glu. Domain analysis using CD-Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and SMART (http://smart.embl-heidelberg.de) revealed that FaNDUFB9, consisting of 117 amino acids, harbours the conserved Complex1_LYR_NDUFB9_LYRM3 domain (Figure 3a). Multiple sequence alignment of FaNDUFB9 with homologous proteins from Fragaria vesca, Rosa chinensis, Pyrus × bretschneideri, Malus domestica, N. benthamiana and Arabidopsis thaliana demonstrated the presence of a highly conserved Complex1_LYR_superfamily motif (Figure 2a). Notably, the amino acid sequences of FaNDUFB9 and FvNDUFB9 were identical, highlighting the high conservation of NDUFB9 in Fragaria × ananassa ‘Benihoppe’ and F. vesca. Phylogenetic analysis further revealed that FaNDUFB9 is closely related to FvNDUFB9 and RcNDUFB9, with progressively distant relationships to NDUFB9 from other Rosaceae members and species from other families (Figure 2b).
FIGURE 2 | Sequence alignment, phylogenetic analysis, expression pattern and subcellular localisation of FaNDUFB9
FIGURE 3 | Mapping functional domains of FaNDUFB9 interacting with SMoV Pro2Glu
SMoV Pro2Glu is a known weak silencing suppressor . To determine whether FaNDUFB9 modulates the silencing suppression activity of Pro2Glu, experiments were conducted using GFP-transgenic N. benthamiana 16c plants . Coding sequences of GFP, FaNDUFB9 and Pro2Glu were cloned into the dual-expression vector pCHF3 and transferred into N. benthamiana 16c plants via Agrobacterium-mediated infiltration to suppress GFP expression. At 5 dpi, GFP fluorescence was observed in plants infiltrated with pCHF3:Pro2Glu/pCHF3:00 and the positive control P19, indicating successful suppression of GFP silencing by Pro2Glu (Figure 4a).
FIGURE 4 | FaNDUFB9 Regulation of Pro2Glu silencing suppression activity
To examine the impact of NDUFB9 expression on Pro2Glu levels, virus-induced gene silencing (VIGS) was employed to silence the homologous NDUFB9 gene in N. benthamiana. By aligning the FaNDUFB9 sequence using the BLAST tool on the Sol Genomics Network (https://solgenomics.net/tools/blast/), the homologous gene NbNDUFB9 was identified. VIGS fragments were then designed using the Sol Genomics Network VIGS tool (https://vigs.solgenomics.net/).Y2H analysis confirmed the interaction between NbNDUFB9 and Pro2Glu (Figure S1). Sequence alignment revealed 73% nucleotide and 78% amino acid similarity between FaNDUFB9 and NbNDUFB9 (Figure S4). A silencing fragment targeting NbNDUFB9 was cloned into the VIGS vector pTRV2. Agrobacterium carrying pTRV1 was mixed with Agrobacterium harbouring pTRV2-NbNDUFB9 and infiltrated into N. benthamiana leaves. Empty pTRV1 and pTRV2 vectors served as controls. RT-qPCR analysis of systemic leaves collected at 5 dpi demonstrated approximately 50% silencing efficiency for NbNDUFB9 (Figure 5a), while expression levels of the other three homologous NbNDUFB9 genes remained unchanged (Figure S5a).
FIGURE 5 | FaNDUFB9 inhibits Pro2Glu expression.
To further assess the effect of FaNDUFB9 on the SMoV, genomic RNA1 and RNA2 of SMoV were transiently expressed via Agrobacterium infiltration in leaves of both 1300-OE and FaNDUFB9-OE plants. Samples were collected at 3 dpi, and RT-qPCR was used to quantify SMoV RNA levels. FaNDUFB9-OE plants exhibited a significant reduction in SMoV genomic RNA compared with control 1300-OE plants (Figure 6). These findings indicate that FaNDUFB9 reduces SMoV RNA levels by interacting with Pro2Glu, underscoring its potential role in mitigating viral activity.
FIGURE 6 | FaNDUFB9 influences SMoV RNA levels.
Related News
2025-07-10
2025-07-08
2025-07-03
Literature Sharing | Regulation of co-translational mRNA decay by PAP and DXO1 in Arabidopsis
2025-07-01
2025-06-27
2025-06-24
2025-06-20
Contact Us
Tel: +86 025-85205672
Email: info@pronetbio.com
Address: Building 3C, Nanjing Xianlin Zhigu,
Qixia District, Nanjing, Jiangsu, China, 210033
Need more info?
Let's connect!