Botrytis cinerea, the causative agent of gray mold disease, is an aggressive fungal pathogen that infects more than 200 plant species. Here, we show that some B. cinerea small RNAs (Bc-sRNAs) can silence Arabidopsis and tomato genes involved in immunity. These Bc-sRNAs hijack the host RNA interference (RNAi) machinery by binding to Arabidopsis Argonaute 1 (AGO1) and selectively silencing host immunity genes. The Arabidopsis ago1 mutant exhibits reduced susceptibility to B. cinerea, and the B. cinerea dcl1 dcl2 double mutant that can no longer produce these Bc-sRNAs displays reduced pathogenicity on Arabidopsis and tomato. Thus, this fungal pathogen transfers “virulent” sRNA effectors into host plant cells to suppress host immunity and achieve infection, which demonstrates a naturally occurring cross-kingdom RNAi as an advanced virulence mechanism.

Aggressive fungal pathogens such as Botrytis and Verticillium spp. cause severe crop losses worldwide. We recently discovered that Botrytis cinerea delivers small RNAs (Bc-sRNAs) into plant cells to silence host immunity genes. Such sRNA effectors are mostly produced by B. cinerea Dicer-like protein 1 (Bc-DCL1) and Bc-DCL2. Here we show that expressing sRNAs that target Bc-DCL1 and Bc-DCL2 in Arabidopsis and tomato silences Bc-DCL genes and attenuates fungal pathogenicity and growth, exemplifying bidirectional cross-kingdom RNAi and sRNA trafficking between plants and fungi. This strategy can be adapted to simultaneously control multiple fungal diseases. We also show that Botrytis can take up external sRNAs and double-stranded RNAs (dsRNAs). Applying sRNAs or dsRNAs that target Botrytis DCL1 and DCL2 genes on the surface of fruits, vegetables, and flowers significantly inhibits gray mold disease. Such pathogen gene-targeting RNAs represent a new generation of environmentally-friendly fungicides.

Pathogens secrete effector proteins to suppress host immune responses. Recently, we showed that an aggressive plant fungal pathogen Botrytis cinerea can also deliver small RNA effectors into host cells to suppress host immunity. B. cinerea sRNAs (Bc-sRNAs) translocate into host plants and hijack the plant RNAi machinery to induce cross-kingdom RNAi of host immune responsive genes. Here, we functionally characterized another Bc-sRNA effector Bc-siR37 that is predicted to target at least 15 Arabidopsis genes, including WRKY transcription factors, receptor-like kinases, and cell wall-modifying enzymes. Upon B. cinerea infection, the expression level of Bc-siR37 was induced, and at least eight predicted Arabidopsis target genes were downregulated. These target genes were also suppressed in the transgenic Arabidopsis plants overexpressing Bc-siR37, which exhibited enhanced disease susceptibility to B. cinerea. Furthermore, the knockout mutants of the Bc-siR37 targets, At-WRKY7, At-PMR6, and At-FEI2, also exhibited enhanced disease susceptibility to B. cinerea, giving further support that these genes indeed play a positive role in plant defense against B. cinerea. Our study demonstrates that analysis of pathogen sRNA effectors can be a useful tool to help identify host immunity genes against the corresponding pathogen.

A never-ending arms race drives coevolution between pathogens and hosts. In plants, pathogen attacks invoke multiple layers of host immune responses. Many pathogens deliver effector proteins into host cells to suppress host immunity, and many plants have evolved resistance proteins to recognize effectors and trigger robust resistance. Here, we discuss findings on noncoding small RNAs (sRNAs) from plants and pathogens, which regulate host immunity and pathogen virulence. Recent discoveries have unveiled the role of noncoding sRNAs from eukaryotic pathogens and bacteria in pathogenicity in both plant and animal hosts. The discovery of fungal sRNAs that are delivered into host cells to suppress plant immunity added sRNAs to the list of pathogen effectors. Similar to protein effector genes, many of these sRNAs are generated from transposable element (TE) regions, which are likely to contribute to rapidly evolving virulence and host adaptation. We also discuss RNA silencing that occurs between organisms.

Eukaryotic regulatory small RNAs (sRNAs) that induce RNA interference (RNAi) are involved in a plethora of biological processes, including host immunity and pathogen virulence. In plants, diverse classes of sRNAs contribute to the regulation of host innate immunity. These immune-regulatory sRNAs operate through distinct RNAi pathways that trigger transcriptional or post-transcriptional gene silencing. Similarly, many pathogen-derived sRNAs also regulate pathogen virulence. Remarkably, the influence of regulatory sRNAs is not limited to the individual organism in which they are generated. It can sometimes extend to interacting species from even different kingdoms. There they trigger gene silencing in the interacting organism, a phenomenon called cross-kingdom RNAi. This is exhibited in advanced pathogens and parasites that produce sRNAs to suppress host immunity. Conversely, in host-induced gene silencing (HIGS), diverse plants are engineered to trigger RNAi against pathogens and pests to confer host resistance. Cross-kingdom RNAi opens up a vastly unexplored area of research on mobile sRNAs in the battlefield between hosts and pathogens.

The exchange of small RNAs (sRNAs) between hosts and pathogens can lead to gene silencing in the recipient organism, a mechanism termed cross-kingdom RNAi (ck-RNAi). While fungal sRNAs promoting virulence are established, the significance of ck-RNAi in distinct plant pathogens is not clear. Here, we describe that sRNAs of the pathogen Hyaloperonospora arabidopsidis, which represents the kingdom of oomycetes and is phylogenetically distant from fungi, employ the host plant’s Argonaute (AGO)/RNA-induced silencing complex for virulence. To demonstrate H. arabidopsidis sRNA (HpasRNA) functionality in ck-RNAi, we designed a novel CRISPR endoribonuclease Csy4/GUS reporter that enabled in situ visualization of HpasRNA-induced target suppression in Arabidopsis. The significant role of HpasRNAs together with AtAGO1 in virulence was revealed in plant atago1 mutants and by transgenic Arabidopsis expressing a short-tandem-target-mimic to block HpasRNAs, that both exhibited enhanced resistance. HpasRNA-targeted plant genes contributed to host immunity, as Arabidopsis gene knockout mutants displayed quantitatively enhanced susceptibility.

Humans, animals, and plants are constantly under attack from pathogens and pests, resulting in severe consequences on global human health and crop production. Small RNA (sRNA)-mediated RNA interference (RNAi) is a conserved regulatory mechanism that is involved in almost all eukaryotic cellular processes, including host immunity and pathogen virulence. Recent evidence supports the significant contribution of sRNAs and RNAi to the communication between hosts and some eukaryotic pathogens, pests, parasites, or symbiotic microorganisms. Mobile silencing signals—most likely sRNAs—are capable of translocating from the host to its interacting organism, and vise versa. In this review, we will provide an overview of sRNA communications between different kingdoms, with a primary focus on the advances in plant-pathogen interaction systems.

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