Current progress on pathogenicity-related genes in Fusarium oxysporum f. sp. cubense tropical race 4

Abstract

Vascular wilt, a disease caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), is highly destructive to bananas. Identifying genes that contribute to the fungus's virulence is crucial for understanding its pathogenesis. In this review, we provide an overview of recent research on genes involved in various aspects of Foc TR4's pathogenic process. These include signal recognition and transduction, the formation of cellular structures, regulation through microRNA and epigenetic mechanisms, effector secretion, and toxin secretion. We place a particular emphasis on discussing effectors that either facilitate virulence or serve as elicitors of host defense responses. Given the limited understanding of the molecular mechanisms underlying Foc TR4 pathogenesis, summarizing the research on these functional genes is necessary and timely. Our integrative information will facilitate research on identification of more key genes involved in the invasiveness of Foc TR4, contributing to more systemic understanding of pathogenesis of this important pathogen. These findings will, in turn, offer potential targets for the development of effective fungicides or soil disinfectants to combat this devastating disease.

Background

Musa spp. (bananas and plantains) is one of the most important fruit crops in the world, serving not only as a dessert fruit but also staple food to millions of people from subtropical and tropical countries (Butler 2013; Wang et al. 2024). The most important cultivars are triploids with an AAA, AAB, or ABB genome constitution, which evolve from diploid wild pointed-leaf bananas (Musa acuminata, AA group) and long-stemmed bananas (Musa balbisiana, BB group) through specific intra- and inter- hybridization. However, the annual production and the quality of banana are greatly reduced by the Fusarium wilt, one of the devastating diseases caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc). According to the susceptibility of banana cultivars, Foc was divided into four physiological races: race 1 infects bananas of the genome groups AAB and ABB and is responsible for the epidemics on the cultivar Gros Michel in the 1950s. Race 2 is only compatible to cooking bananas cv. Bluggoe (ABB). Race 3 only infects wild bananas (Heliconia spp.) but fails to threat cultivated banana varieties. Race 4 infects the Cavendish cultivars (AAA) as well as all the cultivars attacked by races 1 and 2, and thus is currently considered as the most virulent pathogen of Fusarium wilt. Based on regional characteristics of occurrence and range of adaptation to temperature, Foc4 is divided into two types: subtropical race 4 (ST4) affecting Cavendish production in subtropical areas like Canary Islands, South Africa, Australia, and Taiwan of China, and tropical race4 (TR4) was found in the tropical regions of Southeast Asia and Australia (Bai et al. 2013). Of the reported isolates, Foc TR4 is the most destructive Foc variety, as it attacks almost all banana species and has more transmission routes and stronger pathogenicity (Acuña et al. 2022; Mejías et al. 2023).

Foc TR4 produces three types of asexual spores including macroconidia, microconidia, and chlamydospore in its life cycle, enabling it to disperse and survive. The infection cycle is initiated with germination of conidia and formation of hyphae under suitable environment conditions and the favorable host plants. Fungal hyphae then spread and colonize the surface of root. After that, the fungal hyphae surround the epidermis, followed by invading and colonizing the xylem vessels of root. After successfully infecting banana roots, Foc TR4 grows toward the rhizome and pseudostem to proliferate within the vascular tissue, leading to disruption of water translocation and causing the typical wilt symptoms, which include foliage cholrosis, pseudostem longitudinal splitting, necrosis, and ultimate death of the plants (Wang et al. 2024). Finally, fungal hyphae and spores on the debris of banana plant fall into soil through rains and initiate a new infection cycle (Fig. 1). Infection and colonization of banana plants by the fungus always results in wilting and yellowing of the lower part of leaves and discoloration of rhizomes and necrosis of vascular bundles in pseudostem can be observed in the severely infected banana plants (Guo et al. 2014). As a saprophyte, Foc TR4 can survive in soil for long periods in the form of thick-walled chlamydospores (Pietro et al. 2003). Once it perceives the presence of host plants, it begins infecting them from roots (Fig. 1).

Fig. 1

Symptoms of banana vascular wilt and characteristics of the pathogen Fusarium oxysporum f. sp. cubense tropical race 4. a, b Symptom of the Fusarium wilt on the whole banana plant in field. c, d Symptom of the vascular infected by Foc TR4. e Colony growth of the fungus Foc TR4 on the PDA plate cultured for 72 h. f Macroconidia and microconidia produced by Foc TR4. The macroconidium and microconidium are indicated with a black arrow and a white arrow, respectively. g Vegetative hyphae of Foc TR4. h Chlamydospores (indicated by the white arrow) produced by Foc TR4 develop to hyphae (indicated by the black arrow). Scale bars: 10 μm

Due to the above characteristics of colonization and survival, fungicides are largely ineffective to Foc TR4 (Pietro et al. 2003). Since triploid varieties exhibit high sterility, it is also very difficult to develop wilt resistant banana cultivars by traditional breeding method. To date, few effective solutions have been applied to manage this destructive pathogen. Therefore, it is of great urgency and needs to formulate effective control methods for Fusarium wilt of bananas, and this largely requires better understanding of the molecular mechanism underlying Foc TR4 pathogenesis. In the past decades, a few techniques including genomic, transcriptomic, and proteomic methods have been applied to investigate the pathogenesis of Foc TR4. Amongst, as with the sequencing of genomes of the Foc TR4 strain II5, dozens of functional genes have been identified. This review focuses on the recent advances of pathogenic genes of Foc TR4 as well as their roles in pathogenesis and interaction with host bananas.

Genes involved in signaling recognition and transduction

The first step of Foc TR4 in infecting a host is to perceive the presence of host plants, and then to express genes related to the formation of infection structures, in order to successfully infect the plants. Mitogen-activated protein (MAP) kinase cascades have been known to play crucial roles in transducing various extracellular signals and regulating various cellular processes including growth, differentiation, and innate immunity in fungi (Zhao et al. 2005; Johnson et al. 2002; Wu et al. 2024). By homologues sequence analysis with other fungal organisms, the MAP kinase cascade including FocSlt2 (MAPK), FocMkk2 (MAPKK), and FocBck1 (MAPKKK) are identified in Foc. The other two MAPKs are Fmk1 and Hog1 (Wang et al. 2023a) in Fusarium oxysporum but have not been reported in Foc. The null mutants ΔFocslt2, ΔFocmkk2, and ΔFocbck1 all exhibit flexuous hyphal structures and more branches compared to the wild type (WT). The three mutants show similar morphology of hyphae and colony growth, which are all significantly slower than WT. Additionally, FocSlt2, FocMkk2, and FocBck1 are all important for cell wall integrity and hydrogen peroxide response but are dispensable for osmotic pressure response. Compared with the WT control, the ΔFocslt2, ΔFocmkk2, and ΔFocbck1 mutants have less chitin content. Moreover, five out of seven chitin synthases show significantly reduced expression in the three deletion mutants. Additionally, the three kinases are involved in beauvericin biosynthesis by regulating transcription of related genes. The fusaric acid production is significantly reduced in the three mutants by high performance liquid chromatography (HPLC) examination, which is consistent with the observation of lower expression levels of the fusaric acid biosynthesis genes in the mutants compared with WT. Importantly, FocSlt2, FocMkk2, and FocBck1 are all required for full virulence on banana plants. In conclusion, the three mutants by abolishing the MAP kinase-encoding genes show almost identical phenotypes and play vital roles in various biological processes and virulence of Foc TR4 (Ding et al. 2015) (Table 1 and Fig. 2).

Table 1 Genes involved in virulence of Foc TR4

Fig. 2

Schematic representation of proteins involved in different signaling pathways and cellular processes associated with Foc TR4 pathogenicity

Several components of heterotrimeric G-proteins, including Gα, Gβ, and Gγ, have been known to function upstream of the MAP kinase pathway in many fungi (Li et al. 2012). In Foc, the Gα subunit Fga2 and Gβ subunit Fgb1 are also characterized. Phenotypic analyses of the single deletion mutant Δfga2 or Δfgb1 and the double deletion mutant Δfga2/Δfgb1 show that FGB1 but not FGA2 is involved in regulation of colony morphogenesis and conidia production. In addition, the Δfga2, Δfgb1, and Δfga2/Δfgb1 strains produce more pigments than the WT on potato dextrose agar (PDA) plates, suggesting that both FGA2 and FGB1 negatively regulate pigmentation. Both FGA2 and FGB1 have a slight effect on vegetative growth of Foc. Deletion of FGA2 or FGB1 enhances heat resistance in Foc. Notably, the intracellular cAMP levels of Δfga2, Δfgb1, and Δfga2/Δfgb1 are less than 50% of the WT, suggesting that FGA2 and FGB1 are involved in the cAMP pathway and positively regulate intracellular cAMP levels. Pathogen inoculation assays show that G-protein Fga2 and Fgb1 are both required for full virulence of Foc TR4 (Guo et al. 2016) (Table 1 and Fig. 2).

Numerous of transcription factors (TFs) are known as downstream components in the MAP kinase pathway. A MADS-box transcription factor Rlm1 is first reported to play an indispensable role in cell wall integrity as a target of MAPK Mpk1 (Slt2 ortholog) in S. cerevisiae (Dodou and Treisman 1997). FocRlm1 is identified to be highly conserved among Fusarium species. Deletion of FocRLM1 leads to reduced aerial hyphal growth and decreased fungal biomass. The FocRLM1 null mutant exhibits hypersensitivity to H₂O₂, and additionally, by controlling the expression of four peroxidase synthase genes, FocRlm1 is able to regulate anti-oxidation of Foc TR4. Moreover, FocRlm1 is responsible for maintaining cell wall structures of Foc TR4. Notably, FocRlm1 regulates the transcription of fusaric acid biosynthetic genes as well as beauvericin biosynthetic genes. In summary, FocRlm1 is required for full virulence of Foc TR4, and this may result from regulating the expression of genes from RNA-seq of the FocRLM1 deletion mutant compared to WT (Ding et al. 2020). In filamentous fungi, the MAPK kinases, Foc Slt2 (Ding et al. 2015), F. graminearum Mgv1 (Slt2 ortholog), (Hou et al. 2002) and M. oryzae Mps1 (Slt2 ortholog) (Xu et al. 1998) play a crucial role in fungal development and virulence. Interestingly, the ΔFocslt2 and ΔFocrlm1 mutants display distinct colony phenotype. Unlike ΔFocslt2, ΔFocrlm1 shows only slightly reduced aerial hyphae growth, but exhibits significantly reduced biomass. These results indicate that FocRlm1 may be one of the putative downstream TFs, which regulates the expression of multiple MAPK-associated genes involved in fungal development in Foc. Another transcription factor Atf1 is a downstream target of Osm1 in M. oryzae for regulating response to ROS (Li et al. 2012). Osm1 is a mitogen-activated protein kinase homologous to Hog1 (Dixon et al. 1999). Atf1 is also identified in Foc. Disruption of FocATF1 shows enhanced sensitivity to H₂O₂ and causes oxidative burst of banana plantlets in early stages of infection, indicating that FocAtf1 plays an important role in regulation of oxidative stress response and host innate immunity. Importantly, FocAtf1 is responsible for pathogenicity of Foc TR4 on banana seedlings (Qi et al. 2013) (Table 1 and Fig. 2).

Genes required for development of infection-related subcellular structures

During the pathogenic process, synthesis of cell wall is necessary for the fungus invading into host cells. The outermost cell wall, consisted by a crosslink of polysaccharides, is crucial for fungal viability and adaptation to surrounding environment. Phosphomannose isomerase is a key enzyme involved in the biosynthesis of GDP-mannose, an important precursor of the fungal cell wall. Two genes encoding phosphomannose isomerase in Foc, the FocPMI1 and FocPMI2, are identified from homologue analysis with S. cerevisiae. FocPMI1, but not FocPMI2, is highly expressed in the conidial germination and the mycelium stage, indicating that it may play an important role in development of Foc. Notably, the FocPMI1 rather than the FocPMI2 deletion mutant is unable to grow in the absence of mannose, further confirming its role in biosynthesis of GDP-mannose. The optimal concentration of exogenous mannose to support growth of ΔFocpmi1 is 5 mM. Interestingly, fructose or other carbon sources are ineffective to ensure vegetative growth of ΔFocpmi1. In addition, FocPmi1 is involved in cell wall integrity and stress tolerance. Transcriptome analyses show that FocPmi1 regulates expression of genes involved in metabolism of carbohydrates, amino acids, and lipids. Importantly, the pathogen inoculation assay shows that Focpmi1 is responsible for infection and virulence on banana seedlings (Usman et al. 2023). The α-1,6-mannosyltransferase OCH1 is identified and characterized from a T-DNA insertion mutant of Foc. Loss of FocOCH1 leads to impaired fungal growth and reduced number of cell wall proteins. FocOCH1 is involved in cell wall integrity of the fungus. In addition, the FocOCH1 null mutant reduces the ability in hyphal attachment and colonization, and finally results in decreased virulence on banana plantlets (Li et al. 2014) (Table 1 and Fig. 2).

Chitin, a microfibrillar β-1,4-linked homopolymer of N-acetylglucosamine (GlcNAc), is one of the main components of fungal cell wall (Lenardon et al. 2010; Kong et al. 2012). Chitin constitutes the main structure of the fungal cell wall and provides the rigidity (Bowman et al., 2006; Latgé 2007). Chitin synthases are key enzymes catalyzing the polymerization of GlcNAc and are reported to be involved in fungal growth and virulence (Odenbach et al. 2009; Kong et al. 2012). Similar to the model pathogenic fungus Magnaporthe oryzae, there are seven chitin synthases in Foc, the FOIG_07229 (Chitin synthase 1, Chs1), FOIG_10825 (Chs2), FOIG_09216 (Chs3), FOIG_00580 (Chs4), FOIG_06735 (Chs5), FOIG_06738 (Chs6), and FOIG_06723 (Chs7). The expression levels of them have been considered as a standard parameter to evaluate various effects on cell wall biosynthesis (Ding et al. 2020) (Table 2 and Fig. 2).

Table 2 Genes related to several key biosynthetic pathways

Membrane lipids, which are arranged continuously or as a double layer, construct the main skeleton of membranes within the cell, and are crucial for transportation of nutrients and maintenance of osmotic pressure. Glucosylceramides are a class of membrane lipids that serve as vital structural and signaling molecules in eukaryotes. A glucosylceramide synthase named FocGCS has been identified in Foc TR4. FocGCS is highly expressed in germinating conidia and the early infection stage of Foc TR4. The FocGCS null mutant lacks glucosylceramide by sphingolipid profiling analysis. Disruption of FocGCS results in severely abnormal development and substantial loss of virulence. Transcriptome analysis shows FocGCS is closely related to transmembrane transport (Wang et al. 2022a).

Another important subcellular structure required for infection in Foc TR4 is peroxisome. Peroxisomes are single-layer and membrane-bound organelles that are ubiquitously distributed in eukaryotic cells, playing vital roles in β-oxidation of fatty acid to provide cellular energy (Smith and Aitchison 2013). As the marker enzyme of peroxisomes, the peroxidase is a type of oxidoreductases which uses hydrogen peroxide (H₂O₂) as an electron acceptor to catalyze multiple reactions including fatty acid β-oxidation and the glyoxylate cycle, thereby playing an important role in energy metabolism (Chen et al. 2017). Moreover, during interactions of pathogens and host plants, peroxidases catalyze the decomposition of host-derived ROS, which is developed to defend pathogen attacks by plants. In Foc TR4, four genes including FOIG_08821 (PES1), FOIG_07465 (PES2), FOIG_04532 (PES3), and FOIG_09161 (PES4) that are responsible for peroxidase synthesis have been identified. The expression levels of these four peroxidase synthases define the sensitivity of oxidative stress responses (Ding et al. 2020) (Table 2 and Fig. 2).

Key regulators of infection-related genes

Epigenetic regulation plays important roles in the regulation of biological processes, such as growth, development, tissue differentiation, diseases, and interactions between organisms and the environment (Bird 2007; Handel et al. 2010; Verdin et al. 2015). Histone acetylation, catalyzed by histone acetyltransferases (HATs), is one of the main types of epigenetic modification in eukaryotes. FocGcn5 is characterized from homologue sequence blast of Gcn5 in S. cerevisiae and contains a histone acetyltransferase domain Acetyltransf_1, indicating it as a histone acetyltransferase. The null mutant of FocGCN5 shows pleiotropic defects including restricted vegetative growth, dramatically reduced conidiation, and significantly decreased pathogenicity. RNA-seq analysis indicates that the expression levels of around 1500 genes are down-regulated in the FocGCN5 null mutant. Among them, 27 of 29 genes involved in fungal cell wall synthesis, 14 of 16 effector-coding genes, 22 of 23 plant cell wall degrading enzymes-coding genes, and 114 of 137 major facilitator superfamily (MFS) protein-coding genes are confirmed to express at a lower level in ΔFocgcn5 by qRT-PCR analysis. Further analyses demonstrate that FocGCN5 is responsible for environmental stress tolerance and polarity of deposition during cell wall biosynthesis. RNA-seq analysis suggests that FocGcn5 acts as an important regulator of virulence, although it also plays important roles in growth and development (Liu et al. 2022) (Table 1 and Fig. 2).

MicroRNAs (miRNAs), a type of small non-coding single-stranded RNAs primarily found in plants and animals, are known to play crucial roles in diverse biological processes by regulating gene expression. Fungi produce microRNA-like RNAs (milRNAs) that are structurally similar to miRNAs (Sunkar 2010; Ebert et al. 2012). Several proteins including Dicers, QDE2 (Quelling Deficient 2), the exonuclease QIP (QDE2 interacting protein), and MRPL3 (RNAse III domain-containing protein), are involved in milRNAs production (Lee et al. 2010; Chang et al. 2012; Xue et al. 2012). Two Argonaute-coding gene FocQDE2 and FocAGO2, and two Dicer-coding genes FocDCL1 and FocDCL2 are identified in Foc. Phylogenetic analysis shows that FocQde2 and FocAgo2 in Foc are conserved with other Fusarium fungi. Together with FocDCL1 and FocDCL2, the expression of FocQDE2 but not FocAGO2 is significantly up-regulated at 24 h after inoculation on host banana plants, suggesting that sRNAs synthesis mediated by the three proteins including FocQde2, FocDcl1, and FocDcl2 may play an active role during Foc pathogenesis. Phenotypic analyses of deletion mutants showed that FocQDE2, but not FocDCL1 or FocDCL2, is important for colonial morphology. FocQDE2 and FocDCL1 are related to conidia production. Furthermore, FoQDE2 is required for tolerance to oxidative stress. Importantly, the virulence assay indicates that FocQDE2 is required for invasive growth, while FocDCL1 or FocDCL2 is dispensable for full virulence. Based on sRNA sequencing together with molecular methods, a micro-like RNA milR-87 is identified as the target of FocQde2. MilR-87 regulates oxidative tolerance and is involved in pathogenicity of Foc. Further analysis suggests that a glycosyl hydrolase gene (FOIG_15013) is one of the targets of milR-87 and the target site locates at the GH-79C domain in the open reading frame (ORF) region. Moreover, the mutant ΔFOIG_15013 is more virulent than WT, demonstrating it as a negative regulator of Foc pathogenicity. MilR-87 may promote Foc pathogenicity by suppressing FOIG_15013 expression. Meanwhile, further investigation indicates that milR-87 suppresses defense responses likely by silencing the expression of FOIG_15013 (Li et al. 2022) (Table 1 and Fig. 2).

Foc remains as a saprophyte or survives as chlamydospores in the soil in the absence of host bananas, while exists as a pathogen in the presence of banana roots. During this transition, numerous genes related to pathogenicity are required to be reprogrammed, which is governed by several master transcriptional regulators responsible for controlling the gene expression profile. Sge1 (SIX gene expression 1), which was first found in Fusarium oxysporum f. sp. lycopersici (Fol), is one of these regulators capable of regulating expression of effector-encoding genes. The expression of Sge1 is induced in Foc during infection on banana. Phenotypic analyses of the FocSGE1 deletion mutant reveal that FocSge1 affects vegetative growth, conidiation, hydrophobicity, and mycelial pigmentation, probably because FocSge1 is related to the cAMP-PKA signaling since phosphorylation of Sge1 by PKA is required for its function in Fol (Michielse et al. 2009). After 6 weeks of inoculation of banana plantlets, the symptoms including yellowing of the older leaves and cracking of the pseudostem are not observed in the FocSGE1 null mutant-inoculated plants. In comparison, such symptoms are clearly found in banana plantlets inoculated with WT at the third week. This result demonstrates that FocSge1 is required for pathogenicity on banana plants. Moreover, FocSge1 is also related to biosynthesis of fusaric acid (Gurdaswani et al. 2020) (Table 1 and Fig. 2).

Genes encoding secreted proteins and effectors

Foc TR4 invades host plants by secreting a large number of effector proteins to assist in acquiring nutrients, suppressing plant immunity, and thereby promoting its own reproduction and infection. Deciphering the functions of these effector proteins is an important mechanism research of Foc TR4 pathogenesis and has also been a hot topic and frontier research field recently. With protein extraction and secretome analysis, 129 and 105 secretory proteins are found from Foc TR4 cultured alone and cultured with banana roots, respectively. The protein numbers are 120 and 109 from Foc TR1 under the same conditions. Secretory proteins related to hydrolase activity, oxidoreductase activity, and transferase activity are commonly found between Foc TR1 and Foc TR4. Notably, in culturing with banana roots, Foc R1 and Foc TR4 secrete many novel secretory proteins, of which approximately 90% (Foc R1, 57/66; Foc TR4, 50/55) are unconventional secretory proteins which lack signal peptides. In addition, a high proportion of secretory proteins specifically found in Foc TR4 are associated with a number of metabolic pathways such as cysteine and methionine metabolism, carbon metabolism, and phenylalanine metabolism (Wang et al. 2020). Effectors are classified into three categories based on their main roles during the infection.

Effectors for promoting infection or immunity suppression

A cysteine biosynthesis enzyme O-acetylhomoserine (thiol)-lyase (OASTL) is one of the most abundant and inducible Foc TR4-specific secretory protein in presence of plant roots. OASTL is important for pathogenicity but dispensable for vegetative growth of Foc TR4. This is likely because OASTL is responsible for biosynthesis of cysteine, which is the precursor of of the well-known sulfur-containing defense compounds (Wang et al. 2020). The cerato-platanin (CP) protein family was first described and found in Ceratocystis fimbriata, an ascomycete pathogen of the European plane tree (Pazzagli et al. 1999). Since then, CP proteins have been reported in many different filamentous fungi and have also been identified as secreted proteins both in Ascomycota and Basidiomycota. CP proteins are known to be secreted into the culture filtrate, but they are also found in the cell wall of ascospores, conidia, and hyphae (Boddi et al. 2004). Transcriptomic analysis of resistant and susceptible banana roots that are inoculated with Foc TR4 had led to the discovery of plenty of candidate effectors, including a CP protein FocCP1. FocCP1 is highly expressed during the spore germination and infection process. It is localized to plant apoplast and can induce necrosis in banana leaves. Notably, loss of FocCP1 markedly reduces the pathogenicity of Foc TR4 on bananas and restricts invasive growth in banana roots (Liu et al. 2019).

Another novel effector protein FocSP9 has a functional signal peptide but does not contain known motifs or domains. FocSP9 is dispensable for asexual development but required for full virulence by affecting invasive growth in the rhizomes of bananas. RNA-seq analysis show FocSP9 affected the expression of many virulence-associated genes (Guo et al. 2022). FoSsp17 is one of the candidate effectors identified from a streamlined screening system (candidate effector prediction, RNAseq-based expression level analysis, and cell death manipulative activity assessment based on transient expression in N. benthamiana) of Foc. Disruption of FoSSP17 leads to reduced virulence but has little effect on vegetative growth and conidiation. Moreover, FoSsp17 suppresses pattern-triggered immunity in plants by inhibiting reactive oxygen species (ROS) accumulation, reducing callose deposition, and suppressing the expression of NbLOX (associated with the jasmonic acid-pathway) and NbERF1 (related to the ethylene-pathway) (Wang et al. 2023b). FocM35_1, a secreted metalloprotease, was significantly induced during the early stage of Foc TR4 infection. FocM35_1 reduced banana chitinase activity by interacting with the banana chitinase MaChiA. Expression of FocM35_1 could not only facilitate Foc TR4 infection in bananas, but also suppress the INF1-induced hypersensitive response in N. benthamiana. These results indicate that metalloprotease effector FocM35 contributes to pathogen virulence by inhibiting the host immunity (Zhang et al. 2021).

A Cupin type-1 domain-containing protein (named as FoCupin1) was identified as another candidate effector. FoCupin1 has a signal peptide but does not have transmembrane domain or GPI-anchor site. Expression of FoCupin1 is significantly induced at the early stage of Foc TR4 infection on bananas. Importantly, FoCupin1 is required for full virulence of Foc TR4 and shows little effect on fungal growth, conidiation, fusaric acid production, and cell wall integrity. In addition, deletion of FoCupin1 results in activation of multiple defense responses and ROS accumulation in banana root, which restricts fungal penetration and proliferation, and finally decreases virulence of Foc TR4 on bananas (Yan et al. 2022). An exo-polygalacturonase (exo-PG), Pgc4, is purified from a culture of Foc4. The expressed recombinant Pgc4 is capable of causing tissue maceration and necrosis, indicating that Pgc4 may act as a virulence factor in Foc4. Compared with the WT, the PGC4 deletion mutant shows slightly slower hyphal growth and significantly reduced virulence on the banana plantlets (Dong et al. 2020).

Glycosylphosphatidylinositol (GPI)-anchored proteins are important ester modified proteins and are commonly found in eukaryotes. Multiple functions have been reported for the GPI-anchored proteins in filamentous fungi, such as serving as enzymes, cell surface antigens, cell wall proteins, and extracellular matrix proteins. ECM33 is identified as a GPI-anchored protein in Foc TR4. FocECM33 is highly expressed at the early stage of infection on banana roots. Disruption of FocECM33 leads to the defected vegetative growth, the wrinkling hyphae and conidia, and the weakened colonization ability. The FocECM33 deletion mutant shows almost two-folds of endogenous ROS levels as the WT. Three ROS synthesis-related genes all show higher transcription levels in the FocECM33 deletion mutant compared to the WT and the complementary strain. The high endogenous ROS production may cause cell damage of the mycelia in the FocECM33 deletion mutant. Moreover, loss of FocECM33 not only results in increased cell wall sensitivities and chitin synthesis, but also triggers stronger PTI in banana plantlets. Transcriptome analysis reveals that FocECM33 is closely associated with the membrane biological process and chitin accumulation of Foc TR4 (Huang et al. 2022).

F. oxysporum harbors a putative protein with the hallmarks of fungal-pheromone precursors and secretes an-pheromone to act as a growth regulator, chemoattractant, and quorum-sensing signaling molecule. Foc4-PP1, ortholog of the-pheromone precursor gene in TR4 has been identified. Knocking out of Foc4-PP1 leads to slightly lower colony growth and significantly reduced virulence on banana roots. The FocPP1 deletion mutant is more sensitive to Congo red and H₂O₂, suggesting that Foc4-PP1 plays an essential role in cell wall integrity and oxidative response. When transiently expressed in N. benthamiana, Foc4-PP1 is found to localize in both the nucleus and cytoplasm of plants. In addition, Foc4-PP1 and its signal peptide-deficient variant (Foc4-PP1^Δsp) can inhibit BAX-induced cell death, suggesting its role in modulating plant immunity (Liu et al. 2023) (Fig. 2).

SIX effectors

Unlike foliage pathogens, Foc TR4 causes typical symptoms in xylem. It is a good solution to seek secreted pathogenic proteins in xylem of host plants after invasion of this pathogen. “Secreted In Xylem” (SIX) genes were first identified in F. oxysporum f. sp. lycopersici (Fol) with proteomic analysis in xylem sap (Rep et al. 2002). A total of 15 SIX genes have been identified in all F. oxysporum formae speciales (Rep et al. 2004; Houterman et al. 2007; Lievens et al. 2009; Ma et al. 2010; Rep and Kistler 2010; Thatcher et al. 2012; Schmidt et al. 2013; Ma et al. 2015; Williams et al. 2016; Czislowski et al. 2018; Simbaqueba et al. 2018). The protein sequences of SIXs display little homology with each other or known proteins, and the biological functions of SIXs remain largely unknown (An et al. 2019). In Foc, seven SIX genes, the SIX1, SIX2, SIX6, SIX7, SIX8, SIX9, and SIX13, have been identified based on homologous sequence alignment with Fol. Among these genes, SIX2 and SIX8 have only been detected in Foc race 4. SIX8 has been proved to be a functional gene which distinguishes race 4 from race 1 and 2 isolates. Moreover, SNP variations of SIX8 can further differentiate tropical and subtropical race 4 isolates (Fraser-Smith et al., 2014). In Foc TR4, functions of several SIX genes have been clarified. SIX8 can be secreted to the extracellular space and is required for virulence. In comparison, SIX2 is dispensable for pathogenicity since its deletion mutant causes similar disease symptom as WT when inoculating the banana plantlets (An et al. 2019). In Focub II5 genome, three homologues of Fol-SIX1 are annotated as SIX1a, SIX1b, and SIX1c. These genes show over 80% nucleotide identity to Fol SIX1, and the corresponding proteins exhibit around 70% amino acid identity to Fol Six1. Knocking out of SIX1a in FocTR4 severely reduced its virulence to Cavendish banana. The role of SIX1b and SIX1c in FocTR4 virulence remains to be investigated (Widinugraheni et al. 2018) (Fig. 2).

Plant defense elicitors

Foc4 secrets a type of proteins called elicitors which activate plant immunity. A small secreted protein FocSsp1 is proven to be a putative elicitor that negatively regulates pathogenicity of Foc4. Knocking out of FocSSP1 leads to increased virulence and conidiation. In addition, expressions of four pathogenesis-related genes (PR1, PR3, PR5, and PR10) of bananas that are infected with the FocSSP1 deletion mutant are down-regulated in comparison with that of WT. Moreover, PR1b and LOX which are known to be related to the salicylic acid signaling and the jasmonic acid signaling respectively, are both significantly induced at 48 h after inoculation with the FocSSP1 null mutant on N. benthamiana. These results show that FocSsp1 is likely to be an elicitor which triggers plant defense response (Wang et al. 2022b). Another plant defense elicitor is the Fusarium special effector 1 (Fse1). It is significantly induced after colonizing the banana root in Foc TR4. FSE1 is not required for vegetative growth and conidiation. Disruption of FSE1 increases the disease index, while overexpression of FSE1 decreases virulence on banana plantlets. Additionally, Fse1 interacts with a MaEFM-like MYB transcription factor in plant nucleus, and can induce cell death in tobacco leaves (Yang et al. 2023).

Toxins

Fusaric acid (FSA) is a phytotoxin produced by several Fusarium species and known to show strong phytotoxicity on plants and to facilitate infection (Fakhouri et al. 2003; Liu et al. 2020). In Foc TR4, FSA is also proven to be associated with plant disease development (Liu et al. 2020). Similar to other Fusarium species including F. verticillioides and F. fujikuroi, a total of 12 genes designated as FUB1–12 have been identified in the fusaric acid biosynthetic (FUB) cluster in Foc TR4 (Brown et al. 2015; Liu et al. 2020). Of the twelve genes, FUB1, FUB2, FUB3, FUB4, FUB5, and FUB10 have been successfully knocked out and are all found to be critical for FSA production in Foc TR4. Moreover, the pathogen inoculation assay shows that pretreatment with FSA promotes Foc TR4 invasion. In addition, FSA inhibits mitochondria function by regulating the expression of related genes and facilitates disease development by inhibiting the accumulation of host-derived ROS. In conclusion, FSA functions as a positive virulence factor of Foc TR4 (Liu et al. 2020) (Table 2 and Fig. 2).

Beauvericin (BEA) is a cyclohexadepsipeptide mycotoxin produced by many fungi such as Beaveria bassiana and Fusarium spp. (Wang et al. 2012). BEA exhibits various bioactivities including insecticidal, anti-tumor, and anti-microbial activity. BEA has been reported to be produced by Foc. Three BEA biosynthetic genes, FOIG_15793 (BEAS ortholog), FOIG_15792 (KIVR ortholog), and FOIG_15791 (ABC3 ortholog), have been identified according to orthologues analysis of the BEA biosynthetic genes in F. oxysporum f. sp lycopersici race 2 (Lopez-Berges et al. 2013; Ding et al. 2015) (Table 2 and Fig. 2).

Conclusions and perspectives

The Fusarium wilt caused by Fusarium oxysporum f. sp. cubense tropical race 4 is the destructive disease in bananas. Identification of functional genes associated with virulence of Foc TR4 is crucial to understand the pathogenesis, and is necessary for further developing effective strategies to protect bananas from the vascular wilt. In this review, we summarize the current knowledge of functional proteins involved in four stages during the pathogenic process including signal recognition and transduction, cellular synthesis, gene expression regulation, effectors, and toxin secretion. Because of the limited information of pathogenesis and functional research of genes, the presented summary will help researchers focus on banana Fusarium wilt and will advance more systemic research on elucidating pathogenesis of Foc TR4. Overall, based on the increasing knowledge on this fungus and advancing method such as omics and gene editing, a variety of Foc TR4 pathogenicity-related genes will be identified and explored. Research on key enzyme-mediated pathways or posttranslational modifications will be the focus in future. Utilization of these genes as targets to develop specific soil disinfectant or fungicide will be facilitated. In conclusion, more pathogenic genes will be identified to reveal pathogenesis of Foc TR4 and to be utilized to control this devastating disease.