Fungal plant pathogens use proteinaceous effectors as well as newly identified secondary metabolites (SMs) and small non-coding RNA (sRNA) effectors to manipulate the host plant’s defense system via diverse plant cell compartments, distinct organelles, and many host genes. However, most molecular studies of plant–fungal interactions have focused on secreted effector proteins without exploring the possibly equivalent functions performed by fungal (SMs) and sRNAs, which are collectively known as “non-proteinaceous effectors”. Fungal SMs have been shown to be generated throughout the plant colonization process, particularly in the early biotrophic stages of infection. The fungal repertoire of non-proteinaceous effectors has been broadened by the discovery of fungal sRNAs that specifically target plant genes involved in resistance and defense responses. Many RNAs, particularly sRNAs involved in gene silencing, have been shown to transmit bidirectionally between fungal pathogens and their hosts.
Plants have developed a broad spectrum of responses to counter pathogen invasion. Likewise, plant pathogens orchestrate a highly calibrated array of pathogenicity strategies in their quest to cause diseases [1]. The recent increased availability of fungal and plant genomes in the public domain has facilitated considerable progress in molecular plant–fungal interaction studies. Using genetic techniques, pathogenicity or virulence factors have been established, and the study of these factors has increased our understanding of the interactions between pathogens and their hosts. During interaction with their hosts, fungal plant pathogens secrete many proteins known as effectors which manipulate the physiology of the host or suppress the host’s immunity to promote infection [2][3]. Most studies on effectors have focused almost exclusively on secreted proteins, without exploring the possibly equivalent functions performed by fungal secondary metabolites (SMs) (chemical effectors) and sRNAs (sRNA effectors) which are collectively referred to as non-proteinaceous effectors [2][4]. Accumulating evidence has indicated that, pathogens use sRNAs (such as siRNAs and microRNAs) and SMs to manipulate host cell functions [5][6][7][8][9]. Fungal SMs and sRNAs have been shown to manipulate host defense-related genes in the same was as proteinaceous effectors [7][810][11]. In general, SM and sRNA effectors are increasingly becoming important targets for studying the pathogenesis mechanisms of fungal pathogens [911]. Furthermore, these recently discovered SM and sRNA effector entities have been shown in a number of studies to be essential in manipulating host immunity and defense-related genes [2]. It is thus important to adopt new experimental methods to elucidate the in-planta biology of SM and sRNA effectors.
Fungal SMs are not required for the growth and development of the fungus, but they have the potential to improve the pathogen’s fitness under certain conditions. Fungal SMs are often divided into polyketides, terpenes, non-ribosomal peptides and alkaloids on the basis of the primary enzymes and precursors that are involved in their biosynthesis [1012][13][1114]. They eplay a role prior to disease by shaping the plant microbial community, allowing producers to be fully adapted. The existence of fungal SMs, which have no discernible effect on the viability of the producer, raises issues about their potential influence on the environment [1215]. SMs production by fungal pathogens and the presence of a host protein that is specifically susceptible to the corresponding toxin determines the ability of the pathogen to infect the host plant. Because host-specific toxin targets are encoded by plant genes, such genes can be referred to as dominant susceptibility genes [1316]. It is generally known that mutualistic or pathogenic interactions between plants and fungal pathogens entail the simultaneous generation of molecular signals [10][17].
Accumulating evidence indicates that fungal SMs serve as avirulence factors, host defense suppressors, and fungal cell wall hardening factors [810][1418]. Fungal SMs are most effective during the early stage of infection (biotrophic phase), enhancing the fungus’ ability to penetrate and colonize its host without killing its host [2]. Fungal SMs can be host specific or non-host specific and generate necrosis in plant tissue. However, some fungal SMs have functions linked to virulence that are not related to necrosis [1][19][20]. As long as the host plant has the relevant molecular target, such as a resistance gene product, SMs serving as host-specific effectors are thought to play an important role in pathogen virulence [1][21]. Paradoxically, SMs acting as non-host specific effectors have been widely regarded as critical components of pathogen arsenals, despite the fact that they may not be required for pathogenesis [22]. The majority of the fungal SMs have not been defined chemically, and the plants that they are intended to affect are still a mystery. The biological actions that have been reported to be caused by fungal SMs generated in-planta suggest that they have a broad range of plant cellular targets. Some fungi use high affinity iron chelator siderophores synthesized by NRPSs to scavenge environmental iron or to sequester cellular reactive iron [23][24]. These production and transiderophores are essential for fungal growth and development, thus enhancing pathogenicity of various fungal pathogens. Cytochalasans, a diverse group of fungal PKS-NRPS hybrid metabolites, inhibit actin polymerization [25]. The production and transport of proteins are targets of a wide range of fungal SMs [26][1527]. For example, the mycotoxin deoxynivalenol (DON), a member of the type B trichothecenes, produced by Fusarium spp., inhibits protein biosynthesis by binding to the ribosome, resulting in cell signaling, differentiation, reproduction and even teratogenicity disorders in eukaryotes [28][29][30]. A comparative transcriptome analysis of symptomatic and symptomless wheat tissues revealed a substantial induction of TRI genes in symptomless tissues, indicating that DON plays an important role in modulating host defenses and infection establishment [31]. Metabolite profiling of F. graminearum wild-type and the tri5 deletion mutant in infected rachis nodes supports the function of DON in suppressing host defense-related metabolites [32]. DON was demonstrated to modulate programmed cell death (PCD) of host plant cells in a concentration-dependent way [33][34]. A higher concentration of DON may be produced by F. graminearum during infection to trigger hydrogen peroxide (H2O2) production by increasing the size of the hyphal colony. This results in further induction of PCD in wheat [33][34], and thus enhances its switch from biotrophy to necrotrophy [32]. Therefore, it can be unequivocally concluded that, during infection, the mycotoxin DON is produced as a sophisticated strategy of the fungal pathogen to circumvent and hijack the host plant’s defense system.
Cochliobolus species were reported to produce host-specific toxins that enhance pathogen virulence. Victorin, a non-ribosomal peptide produced by Cochliobolus victoriae, is a virulence factor that enhances pathogenicity by inducing PCD during infection of only oat cultivars harboring susceptible genes [35][36][37]. It was also reported that victorin targets the plasma membrane and triggers PCD signaling pathways. HC-toxin, a non-ribosomal peptide produced by Cochliobolus carbonum induces histone hyperacetylation through the inhibition of histone deacetylases, during the infection of only maize varieties harboring susceptible genes [38][39]. Transcriptional activation of host plant defense genes is altered by such histone modifications, thereby enhancing pathogen virulence [39][40]. Alternaria alternata have various pathotypes that produce different host specific toxins that are active only on their corresponding susceptible hosts [41]. Some host specific toxins including destruxin which is produced by Alternaria brassicae are also essential for pathogens in susceptible host plants. A. alternata also produces AAL-toxin, an SM which enhances pathogenicity in tomato varieties harboring susceptible genes by inhibiting ceramide synthase. This will lead to free phytosphingosine and sphinganine accumulation followed by the disruption of plasma membrane [42]. Depudecin is another SM produced by Alternaria brassicicola which also enhances pathogenicity by inhibiting histone deacetylases; its role in pathogenicity is weaker than that of HC-toxin [43]. Tenuazonic acid produced by members of the genus Alternaria and other phytopathogenic fungi inhibits protein biosynthesis on ribosomes [44][45]. It was hypothesized that the Colletotrichum graminicola disease cycle is supported by monorden and monocillins in various ways, initially promoting biotrophic asymptomatic infection by inhibiting Hsp90 chaperons of R-proteins, and disrupting a maize hypersensitive response by enabling a switch to necrotrophy through suppression of basal plant defenses [46]. Fungal SMs such as ophiobolin and herbarumin enhance virulence by inhibiting calmodulin signaling which will disrupt plant regulatory networks [47][48]. It was also demonstrated that Colletotrichum higginsianin SM higginsianin B inhibits jasmonate-mediated plant defenses [49]. Two non-ribosomal octapeptides Fusaoctaxin A and B, which are biosynthesized by the gene cluster fg3_54, were found to be F. graminearum virulence factors [50][51]. Fusaoctaxin A alters the subcellular localization of chloroplasts in coleoptile cells and inhibits callose deposition in plasmodesmata during pathogen infection, thereby facilitating F. graminearum cell-to-cell penetration in wheat cells [50][1651].
sRNA | sRNA Origin | Target Origin | Target Genes | Function | Reference |
---|---|---|---|---|---|
miR408 | Puccinia striiformis f. sp. tritici (Pst) | T. aestivum | CLP1 | Negatively regulates host immune response by suppressing the expression of CLP1. | [2262] |
Pst-milR1 | Pst | T. aestivum | PR2 | Represses plant innate immune response by suppressing the expression of PR2. | [7] |
Pst-milR1 | Pst | T. aestivum | SM638 | Innate immunity. | [7] |
pt-mil-RNA1 | Pt | T. aestivum | TCP14, CYB5R, and EF2 | Suppresses wheat defense response to Pt by targeting wheat TCP14, CYB5R and EF2. | [189] |
pt-mil-RNA2 | Pt | T. aestivum | TCP14, CYB5R and EF2 | Suppresses wheat defense response to Pt by targeting wheat TCP14, CYB5R and EF2. | [189] |
miR398 | Bgh | Barley | HvSOD1 | Negatively regulates host immunity by repressing HvSOD1 accumulation. | [2363] |
miR9836 | Bgh | Barley | MLA1 | Dampens immune response signaling triggered by host MLA immune receptors. | [2464] |
Fg-sRNA1 | F. graminearum | Chinese spring wheat | TaCEBiP | Suppresses wheat defense response by targeting and silencing TaCEBiP. | [2560] |
Fol-milR1 | Fusarium oxysporum | Tomato | SlyFRG4 | Suppresses host immunity by silencing SylFRG4. | [2661] |
Osa-miR167d | M. oryzae | Rice | ARF12, WRKY45 | Negatively regulates host immunity by downregulating AR12 expression. | [2765] |
miR156 | M. oryzae | Rice | SPL14 | Enhances host susceptibility by suppressing the expression of SPL14 and WRKY45. | [2866] |
Osa-miR164a | M. oryzae | Rice | OsNAC60 | Negatively regulates host immunity by suppressing OsNAC60 expression. | [2967] |
miR168 | M. oryzae | Rice | AGO1 | Negatively regulates host immunity by suppressing AGO1 expression. | [3068] |
Osa-miR169 | M. oryzae | Rice | NF-YAs | Enhances host susceptibility by suppressing the expression of nuclear factor N-Y (NF-YA) genes. | [3169] |
miR319 | M. oryzae | Rice | TCP21 | Negatively regulates host immunity by suppressing TCP21 expression. | [3270] |
miR396 | M. oryzae | Rice | OsGRFs | Negatively regulates host immunity by suppressing the expression of OsGRFs. | [3371] |
Osa-miR439 | M. oryzae | Rice | Predicted target genes LOC_Os01g23940, LOC_Os01g36270, LOC_Os01g26340 and LOC_Os06g19250 | Enhances host susceptibility by suppressing the expression of predicted target genes LOC_Os01g23940, LOC_Os01g36270, LOC_Os01g26340 and LOC_Os06g19250. | [3472][3573] |
miR444b.2 | M. oryzae | Rice | MADS-box family genes | Negatively regulates host immunity by suppressing the expression of MADS-box family genes. | [3674] |
siR109944 | Rhizoctonia solani | Rice | FBL55 | Suppresses host immunity to sheath blight. | [3775] |
Bc-siR3.2 | Botrytis cinerea (B. cinerea) | A. thaliana | MPK1, MPK2 | Suppresses MPK1, MPK2 function in plant immunity. | [3858] |
Bc-siR3.1 | B. cinerea | A. thaliana | PRXIIF | Suppresses PRXIIF genes. | [3858] |
Bc-siR3.2 | B. cinerea | Solanum lycopersicum | MAPKKK4 | Suppresses MAPKKK4 function. | [3858] |
Bc-siR5 | B. cinerea | A. thaliana | WAK | Suppression the function WAK genes. | [3858] |
Bc-siR37 | B. cinerea | A. thaliana | WRKY7, PMR6 and FEI2 | Suppresses plant immunity by repressing the expression of WRKY7, PMR6 and FEI2. | [3959] |
In effector biology, the mechanisms through which fungal effectors, particularly sRNA and SM effectors, are transported into cells of the host plant to their targets remain a matter of speculation. Accumulating evidence from preliminary studies suggests that genetic material may be transferred from the host plant to the infecting fungal pathogen cell through exosomal biogenesis pathways [117]. At fungal penetration sites, multivesicular compartments aggregate around fungal haustorial complexes in the host cytoplasm, allowing differentiated vesicle trafficking across the plant-pathogen cellular interface to occur anterogradely, and possibly retrogradely. These multivesicular bodies consist of several intraluminal vesicles, which are discharged extracellularly as exosomes into the paramural region after fusion with the plasma membrane [117]. Multivesicular body-like compartments were reported to be involved in trafficking processes at intercellular channels known as gap junctions, nanotubes, and even the internalization of plasma membrane sections by neighboring cells [124]. siRNA species generated in the host silencing donor were suggested to be transmitted to the fungal recipient through an exocytic/endocytic exchange process at the haustorial interface [117]. Exosomes and plasma membrane-budded microvesicles have both been identified as extracellular vesicles (EVs) that are secreted by plant cells and found in the cells of fungal pathogens [125]. EVs serve as mediators of infection and defenses during plant–fungal pathogen interactions (Figure 1). Active extracellular vesicular transport, passive transport via trans-cell wall diffusion, binding and internalization through membrane-associated receptors, and other trans-membrane pores or channels are all possible sRNA trafficking mechanisms across the plant-fungal interface [81][86][126][127][128][129][130]. A diverse collection of plant sRNAs, including miRNAs and siRNAs, are selectively loaded into the EVs of plant cells [55][129]. It was suggested that fungal sRNAs delivery is facilitated by EVs, similar to the suggested plant-extracellular vesicle-mediated sRNA transport [131]. To test this hypothesis, EVs isolated from various fungal pathogens including F. oxysporum [132][133], F. graminearum [133][134], Z. tritici [135], and Ustilago maydis [136], were established and this laid a foundation for future study of cross-kingdom RNA transport in plant-fungal pathogen interactions [137]. The secreted EVs included a variety of membrane-trafficking proteins and numerous proteins for substrate transport, indicating that EVs might serve an important role in RNA trafficking [127]. Altogether, accumulating evidence points to the idea that fungal EVs are a viable method of transporting pathogen effector proteins, sRNAs and SMs into host plant cells, and their packaging in membrane-bound compartments protects them from degradation by the host enzymes and dilution by water in the plant apoplast.
In effector biology, genomes, transcriptomes, proteomes, and metabolomes are mined to facilitate the discovery of potential effector genes for molecular or cellular biology, biochemistry, and reverse genetics. Future studies need to focus on the development of integrated approaches for the molecular and functional characterization of fungal SM and sRNA effectors during interactions with their host plants. Deletion mutants are usually studied for pathogenicity or symbiosis. The most significant obstacles in the generation of deletion mutants in fungi continue to be transformation and homologous recombination. This strategy has various difficulties when used for SM and sRNA effectors; therefore, new experimental approaches are needed to overcome them. Phylogenetics and comparative genomics analyses should be performed before experimental research because they are particularly informative since the number of fungal genomes and documented SM pathways is increasing. In silico studies may provide novel insights into the organization of conserved gene clusters, as well as their limits and evolutionary history. These kinds of approaches are extremely useful in locating gene clusters that play a role in the production of SMs that have been characterized in other fungal species. They also make it possible to predict the production of compounds that are either identical or related to those produced by specific fungal species. The increased availability of fungal genome sequences and next-generation genomic technologies enables the assessment of SM gene clusters in an individual fungus. RNA-Seq has revolutionized transcriptome profiling and is utilized to study SM gene cluster expression during infection. RNA-Seq can simultaneously quantify transcripts from many organisms, making it ideal for studying plant-pathogen interactions. Manipulating strain-unique SM genes involved in host-specific pathogenicity facilitates plant-fungal pathogen interactions research. Using BGC expression in heterologous hosts such as Saccharomyces cerevisiae or Aspergillus spp. may help to overcome functional redundancy and in-planta detection limitations. SMs from plant pathogenic fungi have primarily been evaluated using phytotoxicity tests. The utilization of chemical genetic screenings may also discover actions against phytohormone signaling pathways and PTI responses. For high-throughput chemical screening, this technique may utilize A. thaliana transgenic lines that express reporter genes in 96-well microplates [138]. Patterns of gene expression and regulation may be used to decipher the complicated bidirectional interaction between pathogen and host cells [90]. Using RNA-seq on both the pathogen and the host is an effective way to examine both sides of this relationship [139][140]. The recently discovered CRISPR Cas13 system can be used to study sRNA effectors, for instance, through the inactivation or localization of fungal sRNAs [141]. Recent studies have shown that extracellular vesicles play significant roles in host defense and pathogen virulence as well as being essential tools for communication between plants and pathogens. To induce the silencing of fungal genes essential for pathogenicity, plant cells secrete extracellular vesicles containing sRNAs into fungal cells. Transmission electron microscopy following ultra-rapid cryofixation showed EVs in Golovinomyces orontii extrahaustorial matrix. EVs produced by apoplastic pathogens may be detected from plant washing solutions [142]. Such fluids likely include plant and fungal EVs, making it difficult to determine their source of origin. To find out if plant pathogen EVs carry RNAs that are functional inside host plant cells, more research must be done, including the biogenesis of EVs and how specific molecules are sorted and directed towards them.