Trans-kingdom sRNA silencing in the prevention and control of crop Fusarium wilt disease

Abstract

The Fusarium oxysporum species complex (FOSC), comprising a multitude of soil borne fungal pathogens, represent a major group of plant invaders that cause vascular wilt diseases of crops worldwide. Although sexual reproduction is nebulous in the FOSC, horizontal gene transfer (also well known as trans-kingdom) may contribute to the observed diversity in pathogenic strains. Concerning the safety and costly process of generating genetically modified crops, a plant-disease management strategy not reliant on transgenic approaches remains the most economic and environmental-friendly option. Here, we highlight the current scenario via beginning with F. oxysporum formae speciales and races as well as caused Fusarium wilt disease in field. Subsequently, based on the mechanisms by which small RNA (sRNA) acts in gene silencing, especially in bidirectionally trans-kingdom sRNA silencing between F. oxysporum and their hosts in the development of disease, we summarize technological breakthroughs like Spray-Induced Gene Silencing (SIGS) technology in the developing and applying to protect crops from Fusarium wilt disease. Given that SIGS has been developed and recently applied in controlling crop Fusarium wilt diseases, we propose a potential prevention and control system to exploit crop Fusarium-vasculature interaction leading to further study trans-kingdom sRNA silencing.

Background

Global crop production is severely challenged by a variety of fungal diseases, of which caused by soil borne fungal pathogenic Fusarium oxysporum species complex (FOSC) is one of the most destructive (Wade 1929; Haware et al. 1978; Smith et al. 1999; Fall et al. 2001). Rather than separate taxa, host-specific strains of F. oxysporum are treated such biologic forms as variants of a single species following assigned to formae speciales (f. sp.). The forma speciales designation had no taxonomic standing but served to facilitate communication among plant pathologists (Snyder and Hansen 1940). Presently, F. oxysporum occurs as pathogenic (plant, animal, and human) and non-pathogenic strains. Following entry of plant root, Fusarium wilt pathogen colonize in the vascular system to destroy plant root-water continuum resulting in wilting symptoms and death of plant eventually (Schäfer 1994).

Since small RNA (sRNA)-mediated RNA interference (RNAi) discovered as a conserved regulatory mechanism in almost all eukaryotes, trans-kingdom RNAi is convinced playing a critical role in host pathogen interactions (Knip et al. 2014; Huang et al. 2019; Liu et al. 2016). Recently, it is discovered that fungus-derived natural trans-kingdom sRNAs are transmitted into plant hosts during infection, functioning as RNA effectors to suppress host immunity. On the other hand, host-derived natural trans-kingdom sRNAs are also confirmed to transmit from host plants into the fungal pathogens (Weiberg et al. 2013; Zhang et al. 2016; Ji et al. 2021). Intriguingly, these discoveries prompt the development of Spray-Induced Gene Silencing (SIGS) for plant disease management. As a non-GMO (genetically modified organisms) alternative to Host-Induced Gene Silencing (HIGS), SIGS attenuates pathogen infection by topical application of dsRNA or sRNA molecules to silence pathogen virulence-related genes (Song et al. 2018; Qiao et al. 2021; Ouyang et al. 2023).

This review synthesizes information from studies of FOSC that pertain to its activities as the cause of field crop Fusarium wilt diseases to highlight important gaps in our knowledge. Important considerations for generating successful SIGS strategies against FOSC will also be emphasized as a powerful and eco-friendly method for crop protection from Fusarium wilt diseases. These include characteristics of effective gene targets and their pathways, and new insights into sRNAs properties impacting on cellular uptake and silencing efficiency. The extensive literatures on the genetic and biochemical interplay between host and FOSC is beyond the scope of this review. The interested reader can find many useful points to this rapidly expanding body of knowledge.

F. oxysporum formae speciales and races

The latest review recorded F. oxysporum formae speciales and races that many scientists still refer to dates back to thirty years (Buxton 1957; Armstrong and Armstrong 1981; Correll et al. 1986). To date, F. oxysporum comprises more than 200 host-specific strains (formae speciales), including plant pathogens, animal pathogens, and nonpathogens, many of which have worldwide distributions (O'Donnell et al. 2009; Geldner 2013). From a practical point of view, pathogenic FOSC can invade perennial and annual plants, including mostly land-based as well as many aquatic plants. F. oxysporum strains lead to either wilts or root/crown rots on economically main field crops (e.g. cotton, banana, and soybean), important market garden crops (e.g. tomato, onion, and melon), grain legumes (e.g. cowpea, chickpea, and faba bean), ornamental crops (e.g. cyclamen, orchids, and gerbera), as well as and even on broom or rapewitchweed (e.g. parasitic plants, and weeds) (Abawi and Lorbeer 1972; Brayford 1996; Olivain and Alabouvette 1999; Gao et al. 2021).

Intriguing individual strains display developing selective pathogenicity to a relative narrow range of host plants, which is defined as a forma speciales. For example, strains responsible for Fusarium wilt of tomato belong to the forma speciales lycopersici. Actually, the concept ‘forma speciales’ is first formulated early to distinguish strains of Puccinia graminis Pers. similar morphological characteristics but different host ranges (Stakman 1913; Edel-Hermann and Lecomte 2019; Favre et al. 2023). Among known forma speciales, more than 100 formae speciales are well documented. The host range of these formae speciales consists of plants belonging to 45 families (Asteraceae, Cucurbitaceae, Fabaceae, and Solanaceae are the most represented) (Poli et al. 2012; Pastrana et al. 2017). More than 30 formae speciales are considered as insufficiently documented. These pathogens isolated from diseased plants and assigned to a forma speciales without confirming their pathogenicity, or, host specificity of these formae speciales are not analyzed yet (Armstrong and Armstrong 1981; Gordon et al. 2015). Furthermore, 58 host plant species in the literature are recorded and described as susceptible to F. oxysporum but whose forma speciales has not been characterized so far. Such a wide geographical distribution and wide host spectrum confirms the adaptability of FOCS to the diverse biotic and abiotic environmental conditions encountered worldwide (Steinberg et al. 2016; Edel-Hermann and Lecomte 2019).

However, continuously increasing diversity in FOCS and within formae speciales may be revealed over time with the new plant genotype derived from breeding. This growth may reflect a worrying trend, as more and more new varieties are described as special varieties related to the resurgence of diseases in market horticultural and ornamental crops, as well as large-scale crops. For example, many formae speciales are well known to be polyphyletic obstructing to identify specific molecular markers (Baayen et al. 2000; Fourie et al. 2009; Koyyappurath et al. 2015). Meanwhile, both soil-borne and endophytic non-pathogenic varieties of F. oxysporum exhibit high genetic variability and are closely related to pathogenic isolates (Edel et al. 2001; Inami et al. 2014; Imazaki and Kadota 2015). For this, desirable tools, such as molecular markers, are urgently developed to detect the presence and activity of pathogenic FOCS isolates, as well as discriminate between races and formae speciales.

Factors influencing wilt diseases caused by F. oxysporum

Pathogenic F. oxysporum strains are responsible for two typical types of symptoms, vascular wilting and rotting in roots (without reaching the vascular system). According to the Committee on Standardization of Common names for plant diseases (http://www.apsnet.org), when pathogens penetrate the host roots to reach the xylem vessels, F. oxysporum causes vascular wilting which colonizes upwards, resulting in progressive yellowing and wilting of the plant, associating with several disease names as Fusarium yellows, Fusarium blight, and Fusarium wilt. Meanwhile, diseases with rotting symptoms are called basal rot, Fusarium stem rot, or crown and root rot (Jarvis and Shoemaker 1978; Olivain and Alabouvette 1999). However, some plants, for example tomato, can be attacked by formae speciales lycopersici and radicis-lycopersici causing vascular wilting and root rot, respectively (Vakalounakis 1996).

Most of the Fusarium forma speciales are considered as hemi-biotrophic plant pathogens, killing host by producing reactive oxygen species (ROS), secreting cell wall degrading enzymes (CWDE), and phytotoxic molecules (Desmond et al. 2008; Fones et al. 2020). During the course of invasion, phytopathogenic fungi are perceived through plant cell surface receptors that recognize pathogen-associated molecular patterns (PAMP), secreted by pathogens or released by the action of lytic enzymes during the interaction with the host. PAMPs (including but not limited phospholipomannan, ß-glucans, mannoproteins, and chitin) further activate the first line of defense well-known as PAMP-triggered immunity (PTI) in plants (Jones and Dangl 2006; Kanyuka and Rudd 2019; Pierzgalski et al. 2021). Phytopathogenic fungi evolve to counteract PTI through the production and secretion of molecules called effectors, which initiate the second line of plant defense called effector-triggered immunity (ETI) through the interaction with host intracellular receptors (Jones and Dangl 2006). In most case, investigation of the function of effectors is impeded by the redundancy of homologous genes, therefore, the most promising alternative is to isolate these effectors to uncover new metabolites and proteins associated with pathogenicity or virulence (Müller et al. 2008).

Fungal virulence is termed as a capacity causing damage to the host masking any obvious phenotype (wilt syndrome). Thus, factors influencing wilt diseases caused by F. oxysporum are any molecule that enhances the pathogenicity of the pathogen by interfering with host particular functions. These virulence factors (VF) can be categorized according to the chemical nature, the acting site, and the interaction style. Basically, VF can be classified into two large groups: low molecular weight metabolites (e.g. PAMPs) and proteins (e.g. effectors) (Kraft 1994; Ibrahim et al. 2021). Moreover, nitrogen-containing metabolites, such as ammonium or glutamine, play as critical signal maintaining the expression level of virulence genes in F. oxysporum (Wong et al. 2008; López-Berges et al. 2010). Specifically, secreted in xylem (SIX), a group of small effector proteins in the xylem secreted by F. oxysporum, are well documented contributing to F. oxysporum virulence. So far, fourteen SIXes have been verified, with no surprise, formae speciales vary in SIX protein profile and respective gene sequence (Rep et al. 2004; Maldonado Bonilla et al. 2018; Adusei-Fosu and Dickinson 2019; Lyons et al. 2019). The main VFs reported to date specially for F. oxysporum f. sp. lycopersici are summarized in Table 1. All listed VF are from species F. oxysporum f. sp. lycopersici, with exception SIX from f. sp. lycopersici, cubense, conglutinans, and melonis respectively, and FoEG1 from f. sp. lycopersici and vasinfectum respectively. Among these VF, PGs, Tomatinase, FoMep1, FoSep1, and FoEG1 are located in the apoplast, FoRnt2, FoAPY, and SIX are translocated into the host cell cytoplasm. However, the functional annotation of these VF (or proteins) is not full addressed yet and it is unknown how they contribute to virulence.

Table 1 Main proteins reported as VF from F. oxysporum f. sp. lycopersici

Trans-kingdom sRNA silencing in Fusarium oxysporum-plant interactions

Small RNAs (sRNAs) are 20–30 nucleotide (nt)-long non-coding RNA molecules, which are divided into two major classes: microRNAs (miRNAs) and small interfering RNAs (siRNAs). Most miRNAs are 18–24 nt long and derived from single-stranded RNA precursors with imperfectly base-paired hairpin structures. sRNAs are universally distributed in all kingdoms of life from bacteria, archaea to various eukaryotic lives. It is well established that sRNAs contributes to almost all eukaryotic cellular processes, which have not ceased to surprise us throughout the last three decades regarding their functional and compositional diversity (Grosshans and Filipowicz 2008; Carthew and Sontheimer 2009; Guo et al. 2019; Chen and Rechavi 2022). Since the first miRNA reported in Caenorhabditis elegans in 1993, researchers have begun to explore the generation mechanism and biological functions of microRNAs, as well as the key role in gene expression regulation (Lee et al. 1993; Reinhart et al. 2000). The Nobel Assembly at Karolinska Institutet awarded the 2024 Nobel Prize in Physiology or Medicine jointly to scientists Victor Ambros and Gary Ruvkun ‘for their discovery of microRNA and its role in post-transcriptional gene regulation’. The groundbreaking discovery reveals an entirely new dimension to gene regulation, meanwhile, inspires the wide application in plant protection.

RNA interference (RNAi, or RNA silencing) mechanisms have been studied comprehensively in different organisms in many biological processes (Hannon 2002; Baulcombe 2004). Bidirectional transmission of sRNAs between host plants and fungal pathogens has been documented to influence host-fungus interactions, which provided direct evidence of natural trans-kingdom RNAi, although fungal RNAi mechanisms still require further investigation (Wen et al. 2023). In virus-induced gene silencing (VIGS), the siRNA specificity determinant is derived from the viral RNA. Meanwhile, emerging body of evidence show that plants and filamentous microorganisms employ RNAi to influence each other, which lead to host induced gene silencing (HIGS) affecting gene expression in fungal. However, filamentous organism-induced gene silencing (FIGS) acts in the opposite direction (Baulcombe 2015).

Recently, several studies illustrated that spraying double-stranded RNAs (dsRNAs)/sRNAs on plant surfaces confers efficient crop protection through targeting essential pathogen genes (Koch et al. 2013, 2016; Wang et al. 2016; Qiao et al. 2021; Ouyang et al. 2023). Thus, a termed spray-induced gene silencing (SIGS) is known as a powerful, fast, sustainable, and environmenta- friendly strategy to circumvent the problems in creating GMOs (Wang and Jin 2017). Previously, we explored the RNAi-based SIGS strategy using direct application of FolRDR1-dsRNA to protect tomato wilt disease caused by F. oxysporum f. sp. lycopersici (Fol). Our data alternatively demonstrated that FolRDR1 mediated the pathogen development and pathogenicity. Both Fol and tomato efficiently took up FolRDR1-dsRNA from the environment through spraying on tomato seedling leaves. Further, exogenous application of FolRDR1-dsRNAs significantly alleviated the progress of tomato wilt disease symptoms. More than intriguing, the fluorescence signals of FolRDR1-dsRNAs were dominantly localized in the host vascular bundles where microconidia spore are produced and disseminated. Employing the vascular bundles as transport corridor, endogenetic hypha spreading to above ground tissues is critical for disease progression for Fol (Ouyang et al. 2023). Similarly, spraying application of 791 nt noncoding CYP3-dsRNA alleviates the growth of pathogen using the agronomically barley—F. graminearum pathosystem. After uptake via the plant vascular system by the pathogen, CYP3-dsRNAs are processed into small interfering (si)RNAs by fungal DICER-LIKE 1 (FgDCL-1), then target three fungal ergosterol biosynthesis genes (CYP51A, CYP51B, CYP51C) in soil-borne pathogen F. graminearum (Koch et al. 2016). These successful SIGS studies show that such specific Fusarium pathogen gene-targeting RNAs represents a new generation of environmentally-friendly fungicides to control vascular wilt disease (Fig. 1).

Fig. 1

SIGS strategy to protect crops from the invasion of fungal pathogens F. oxysporum. Sprayed exogenous dsRNAs/sRNA can be taken directly into Fusarium cells on the plant vascular system or by first passing through the cells of the plant. In this case, the dsRNAs/sRNA are translocated through vascular system before delivery to the fungal hyphae or conidia. Alternatively, the exogenous processed and/or unprocessed dsRNA, as well as sRNA, are taken into the fungal cell either by haustorial feeding, endocytosis, or vesicle trafficking (the certain entry mechanism of dsRNA/sRNA into the fungal cell still to be determined). In Fusarium cell, the fungal RNAi machinery is guided by siRNAs derived from the exogenous dsRNA to mediate particular target gene silencing, finally leading to impair the pathogenicity of Fusarium pathogens. dsRNAs are processed by DCL family to produce siRNAs and subsequently loaded into the AGO complexes to induce targeted gene silencing (Hannon 2002; Baulcombe 2004; Koch et al. 2016; Ouyang et al. 2023). DCL, Dicer-like protein; AGO, Argonaute protein; Con, conidia; Grey dots, dsRNAs/sRNA. Red arrows, the moving direction of pathogens. Blue arrows, the moving direction of dsRNAs/sRNA

Given the ease of design, high specificity, and applicability to diverse pathogens, and environmente-friendly compared with traditional fungicides and creating GMOs, SIGS immediately attracts the attention of researchers as a new plant protection strategy. To serve as an efficient ‘RNA fungicides’, a reasonable duration of efficacy is concerned and desired for the SIGS strategy. The most challenge of the SIGS technology is the relative instability of RNAs in the natural environment. Strikingly, application strategies can be improved by inorganic/organic nanoparticles as carriers to stabilize the RNAs and thus increase the strength and duration of plant protection (Joga et al. 2016; Christiaens et al. 2018; Schwartz et al. 2020; Martinez et al. 2021). However, there are still several questions need to be addressed, (i) what are the optical properties (e.g., length, secondary structure formation, etc.) of dsRNA fragment for nanoparticles? (ii) how to avoid off-target silencing which may lead to biosafety concerns? (iii) what’s the balance of RNA uptake efficiency between host plant and pathogen?

Conclusion: SIGS in F. oxysporum infections—Where do we stand?

Devastating vascular wilt caused by F. oxysporum, curtailing the production of economically important crops worldwide, has become a menace to farmers. The emergence of Spray-Induced Gene Silencing (SIGS) is a native strategy for crop protection utilizing exogenously applied non-coding RNA (double-stranded RNA or sRNA) to specifically reduce pathogenic target gene expression. Once a disease-resistant phenotype has been developed with SIGS against an accurate target, further optimization of the applied RNA sequence may further increase efficacy (Sharath Chandra et al. 2019; Kweon et al. 2022). The efficiency of non-coding sRNA-mediated gene silencing depends on uptake, processing to siRNAs, accessibility of the target region of the gene, and forming RNA-induced silencing complex (RISC) together with a member of the Argonaute (AGO) protein family (Reynolds et al. 2004; Lo Presti et al. 2015; Fakhr et al. 2016).

Since B. cinerea is found to take up exogenous dsRNAs naturally, SIGS technology has been developed for crop protection against fungal pathogens (Wang et al. 2016; Wang and Jin 2017). In December 22, 2023, the U.S. Environmental Protection Agency (EPA) has registered biopesticide products containing the new active ingredient Ledprona (https://www.epa.gov). Ledprona is a new type of pesticide that relies on RNA interference (RNAi) used to protect against disease in plants and animals. Ledprona is a sprayable dsRNA product that targets the Colorado potato beetle (CPB), a major pest of potato crops, to kill the pest by ‘silencing’ the CPB gene needed to produce the PSMB5 protein. This RNAi-based pesticide is the first SIGS-dsRNA pesticide in the world allowed to be used commercially and sprayed on plants (Rodrigues et al. 2021; Pallis et al. 2022). With the examples discussed in this review, it is conceivable that SIGS-based technology can be deeply elucidated and exploited to further understand crop Fusarium wilt disease. Therefore, the interaction of plants and vascular pathogens is an ideal model for the study of trans-kingdom RNAi, which are relatively more diverse and complex than oomycetes, bacteria, and viruses. Comparative pathogen genomics and deep sRNA sequencing based on microbiome projects would promote exploration of key trans-kingdom sRNAs involved in plant—F. oxysporum interactions, providing new efficient targets for crop protection tools such as SIGS.