Fungal Secondary Metabolites and Small RNAs Enhance Pathogenicity during Plant-Fungal Pathogen Interactions

Abstract

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. However, there are no clear functional approaches to study the role of these SM and sRNA effectors. Undoubtedly, fungal SM and sRNA effectors are now a treasured land to seek. Therefore, understanding the role of fungal SM and sRNA effectors may provide insights into the infection process and identification of the interacting host genes that are targeted by these effectors. This review discusses the role of fungal SMs and sRNAs during plant-fungal interactions. It will also focus on the translocation of sRNA effectors across kingdoms, the application of cross-kingdom RNA interference in managing plant diseases and the tools that can be used to predict and study these non-proteinaceous effectors.

Keywords: cross-kingdom RNAi; extracellular vesicles; non-proteinaceous effectors; plant-pathogen interaction; virulence.

Figure 1

Fungal SM effectors. Host-specific toxins include HC-toxin, victorin, higginsianin B, AAL-toxin, Fusaoctaxin A, destruxin, depudecin. Host non-specific toxins include DON, Fumonisin B1, tenuazonic acid, and cercosporin. Adapted from [51,52,53,54].

Figure 2

Cross-kingdom RNAi and vesicle trafficking during plant-fungal pathogen interactions. Fungal and plant sRNAs trigger cross-kingdom RNAi during plant-pathogen interactions. Fungal sRNAs translocate into plant cells and hijack the host plant Argonaute (AGO) protein of the RNAi machinery to suppress host plant immune response. The fungal sRNAs are upregulated upon infection (indicated by green arrow). Host cells also can deliver sRNAs into pathogen cell, either host induced gene silencing (HIGS) sRNAs or endogenous sRNAs, to target virulence genes and other essential pathogen genes. The generation of multivesicular bodies and release of exosomes at the site of pathogen invasion is part of the host penetration resistance pathway. Among other molecules, the putative exosomes contain sRNAs that can target vesicle trafficking components of the pathogen. Exosomes can also inhibit fungal growth and stall further ingress. The production of pathogen-derived sRNAs that may target and silence host genes can be inhibited by this form of host plant immunity. The fungal pathogens also secrete proteinaceous effectors through the haustorium into the host cells to suppress the host immunity genes, thereby causing disease. How fungal pathogens transport proteinaceous effectors and sRNAs into their host cells is still elusive. On the other hand, plants secrete extracellular vesicles to transport host sRNAs into pathogens to silence fungal genes involved in pathogenicity. Passage of host sRNAs through the haustorial cell wall, either active or passive, occurs and once inside the fungal haustorium the silencing molecules trigger RNAi of their mRNA targets, and may act as primers in the fungal silencing pathway, leading to the generation of systemic silencing signals. Cell structures are not drawn to scale.

Figure 3

Mode of action of F. graminearum-secreted pathogenicity factors during F. graminearum-wheat interaction. The sRNA effector Fg-sRNA1 contributes to virulence by silencing wheat defense-related TaCEBiP. Fungal toxin DON inhibits protein biosynthesis by binding to the ribosome. The fungal toxin Fusaoctaxin A changes the subcellular localization of chloroplasts in the coleoptile cells and prevents callose accumulation in plasmodesmata during pathogen infection, facilitating the cell-to-cell invasion of F. graminearum in wheat tissues.

Figure 4

Integrated approaches to elucidate the biological functions of fungal SM and sRNA effectors during plant-fungal pathogen interactions. (a) The most common approach begins with genome mining integrated with in-planta transcriptome analysis (and/or in-planta proteome/metabolome analysis), which gives potential candidate genes to delete and test for a function in plant colonization. Following these functional tests, plant targets are identified. Each of these steps has bottlenecks (text in red), which necessitate the consideration of complementary or alternative options. Improved bioinformatic prediction of gene composition is critical for secondary metabolite gene clusters in particular. In-planta-omics approaches suffer from fungal material dilution in a complex plant sample. Transformation and homologous recombination continue to be the most significant barriers to the generation of deletion mutants in fungi. Infection assays are often sensitive enough to detect only significant contributions to the infection process. (b) Combinatorial genetic validation employing clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 and Cas13 technologies opens new opportunities for studying the biological functions of fungal SM and sRNA effectors. Heterologous production or enhanced in-planta synthesis of fungal secondary metabolites allows their chemical characterization and subsequently determination of their function using tailored bioassays and spatial distribution in infected plant tissue. Adapted from [5].