Host suppression of the novel virulent effector FocSP1 improves banana resistance to Fusarium oxysporum f. sp. cubense tropical race 4

Dear Editor, Banana (Musa spp.), a widely cultivated crop in tropical or subtropical regions, is under severe threat from Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), the causative agent of fusarium wilt. This fungus can survive for over 40 years in the soil in the absence of a host [1], yet remains highly infectious upon contact with banana roots. Once inside the plant, Foc TR4 colonizes and obstructs the vascular bundles, leading to leaf wilting and eventual plant death. During infection, Foc TR4 secretes many effectors that help suppress host immunity and facilitate pathogen infection. Known effectors, such as the SIX (Secreted in Xylem) homologs, FocSIX8, have been shown to be essential for Foc TR4 full virulence [2]. However, a critical gap in our knowledge of novel and potent effectors of Foc TR4 needs to be filled. To address this knowledge gap, we employed bioinformatics and RNA-seq approaches [3] and identified a novel effector from Foc TR4 with a predicted secreted signal peptide (Fig. 1A), designated Foc-secreted protein 1, FocSP1. RT–qPCR analysis revealed that FocSP1 transcript levels were significantly induced after infection (Fig. 1B). To further validate its expression, we constructed FocSP1:GFP-expressing strains under the control of either its native promoter or a constitutive RP27 promoter. Notably, the strain expressing FocSP1:GFP driven by its native promoter showed no detectable GFP signal when grown on potato dextrose agar (PDA) plates, whereas a strong GFP signal was observed in the RP27 promoter strain (Fig. 1C). However, both strains showed strong GFP signals during banana root infection (Fig. 1C), indicating that the expression of FocSP1 was significantly induced in roots regulated by host-derived signals during pathogenesis. Pathogenicity assays showed that the ΔFocSP1 mutant caused only mild necrosis symptoms in the corm, whereas the wild-type (WT) and the complemented strain ΔFocSP1-C induced significantly more severe disease symptoms (Fig. 1D and E). Collectively, these results indicated that FocSP1 is required for full virulence of Foc TR4. The role of FocSP1 in Foc TR4 pathogenicity and banana immunity. (A) A yeast secretion assay to evaluate the secretion functionality of the FocSP1 signal peptide. Yeast YTK12 strain transformed with either the Invertase-∆sp (pSUC2 empty vector containing an invertase without signal peptide, negative control), Mg87sp (supplemented with non-functional Mg87 signal peptide, negative control), FocSP1sp (supplemented with FocSP1 signal peptide), and Avr1bsp (supplemented with Avr1b signal peptide, positive control) were evaluated for growth on CMD-W or YPRAA medium and for invertase activity assay with 2,3,5-triphenyltetrazolium chloride. (B) RT–qPCR analysis of FocSP1 expression during the early infection stages of banana roots inoculated with Foc TR4. Expression levels were normalized to the fungal reference gene FocEF1α. (C) Fluorescence observation of Foc TR4 strain carrying the FocSP1:GFP construct. GFP signals during vegetative growth of Foc TR4 (top) were observed in mycelia and conidia after 6 days of culturing on PDA plates, while GFP signals in infected banana roots (bottom) were detected at 4 dpi. Scale bar = 20 μm. (D) Disease internal phenotypes and (E) distribution of plant disease scores in banana plants inoculated with Foc TR4, ΔFocSP1, and ΔFocSP1-C strains for 5 weeks. (F) Nicotiana benthamiana leaves 3 days after infiltration with Agrobacterium tumefaciens carrying either a GFP empty vector (EV) only, FocSP1:GFP, or ΔspFocSP1:GFP (FocSP1 devoid of its signal peptide). For co-infiltration assays, the same leaf areas were subsequently infiltrated with A. tumefaciens harboring INF1:GFP. Images were captured 3 days after co-infiltration. (G) Western blotting detection of protein expression in N. benthamiana leaves using anti-GFP antibody. (H) RT–qPCR analysis of FocSP1 expression in oeFocSP1 transgenic banana plants and WT plants. (I) Disease internal symptoms and (J) distribution of plant disease scores observed in banana 35 days post-inoculation with Foc TR4. (K) Relative expression levels of FocSP1 in Foc TR4 mycelium-infected banana leaves subjected to treatments with FocSP1-dsRNA and water by using RT–qPCR. (L) Foc TR4-induced necrosis and necrosis area of banana leaves 7 days post-inoculation. The necrosis area was calculated using ImageJ software. (M) Length distribution and abundance of FocSP1-specific siRNAs. (N) Disease symptoms and (O) distribution of disease scores observed in banana plants 35 days post-inoculation with Foc TR4. (P) RT–qPCR analysis of FocSP1 gene in roots of FocSP1-RNAi and WT plants at intervals of 0, 4 and 8 days post-inoculation. (Q) Hierarchy clustering of DEGs across seven different pairwise comparisons among WT, oeFocSP1 (OE), and FocSP1-RNAi (KO) with (*) and without Foc TR4 II5 infection. The asterisk (*) denotes banana plants infected with Foc TR4. Rows in the heat map represent genes and columns indicate different pairwise comparisons. The heatmap displays log₂ fold-change values. According to the scale bar, positive values (represented by darker shading) indicate up-regulated genes, while negative values (represented by lighter shading) indicate down-regulated genes. (R) GO functional annotation of identified DEGs from seven pairwise comparisons. The numbers to the right of the columns represent the count of up-regulated (left) and down-regulated (right) genes, respectively. (S) Yeast two-hybrid (Y2H) assay showing that FocSP1 interacts with MaRBP1. Cotransformants of p53 + pGADT7-T and lam + pGADT7-T were used as positive control and negative control, respectively. SD/-LWAH and SD/-LW represent SD/−leucine-tryptophan–adenine–histidine and SD/−leucine–tryptophan selective medium, respectively. (T) Luciferase complementation imaging assays showing the interactions between FocSP1 and MaRBP1. Tobacco leaves were divided into four parts and infiltrated with Agrobacterium strains harboring FocSP1-nLUC and MaRBP1-cLUC. The following three pairs of constructs were used as negative controls: FocSP1-nLUC + cLUC, nLUC + MaRBP1-cLUC and nLUC + cLUC. The images were captured at 3 days post-infiltration. Quantitative data are presented as means ± SDs (n = 3). Significance analysis in (B) and (H) was performed using one-way ANOVA through Tukey’s test. Significance analysis in (J) and (O) was performed using the Kruskal–Wallis test. Significance analysis in (K), (L), and (P) was performed using Student’s t-test (**P < 0.01; ***P < 0.001; ns, not significant). To investigate the mechanistic role of FocSP1 in plant immunity, FocSP1 and INF1 were transiently expressed in Nicotiana benthamiana leaves. A GFP-only vector and the Phytophthora infestans cell death elicitor INF1 [4] were used as negative and positive controls, respectively. As expected, apparent cell death in N. benthamiana leaves was induced by INF1 but not FocSP1 (Fig. 1F). Interestingly, co-expression of FocSP1 with INF1 suppressed the cell death typically caused by INF1, while co-expression with the GFP EV had no effect (Fig. 1F). Furthermore, we also found that ΔspFocSP1 (FocSP1 without signal peptide) had the same function as FocSP1 in N. benthamiana leaves (Fig. 1F). Immunoblotting analysis confirmed equivalent protein accumulation in N. benthamiana leaves (Fig. 1G). Taken together, these data suggested that FocSP1 inhibits plant immune responses to facilitate Foc TR4 infection. To further investigate the biological function of FocSP1, we generated two transgenic banana lines (oeFocSP1), both with a high FocSP1 expression level (Fig. 1H). Upon Foc TR4 infection, the oeFocSP1 plants exhibited significantly more severe disease symptoms compared with the WT plants (Fig. 1I and J), suggesting that FocSP1 enhances Foc TR4 infection. Given the compromised virulence of the FocSP1 knockout mutant and the enhanced immunity suppression of FocSP1-expressing plants, we employed a host-induced gene silencing (HIGS) strategy to disrupt FocSP1 [5]. Firstly, we tested the efficacy of a double-stranded RNA (FocSP1-dsRNA) targeting FocSP1 by applying it to banana leaves. The treatment effectively suppressed FocSP1 expression (Fig. 1K) and significantly reduced Foc TR4-induced necrosis (Fig. 1L). To test the impact of in planta suppression of FocSP1 expression on Foc TR4 infection, FocSP1-RNAi transgenic lines were generated using the same silencing sequences. Small RNA sequencing confirmed the presence of FocSP1-specific siRNAs predominantly ranging from 18 to 25 nt in length, with 20-, 21-, and 22-nt small RNAs being the most abundant (Fig. 1M), indicating the expected functionality of the RNAi construct. Subsequently, the susceptibility of FocSP1-RNAi transgenic plants against Foc TR4 infection was evaluated. Following inoculation, 100% of WT plants showed severe disease symptoms, while only ~33.3% plants showed severe disease symptoms. The majority of FocSP1-RNAi plants exhibited mild or no symptoms (Fig. 1N and O). During the infection process, the relative transcript levels of FocSP1 in FocSP1-RNAi plants were reduced to