Fungal small RNAs suppress plant immunity by hijacking host RNA interference pathways

Abstract

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.

Fig. 1. Bc-sRNAs silence host target genes in both Arabidopsis and S. lycopersicum during B. cinerea infection

(A) Bc-siR3.1, Bc-siR3.2, and Bc-siR5 were expressed during infection of Arabidopsis as detected at 18, 24, 48, and 72 hours after inoculation and (B) S. lycopersicum leaves at 18, 24, 32, 48 hours after inoculation by means of reverse transcription polymerase chain reaction (RT-PCR). Actin genes of B. cinerea, Arabidopsis, and S. lycopersicum were used as internal controls. Similar results were obtained from three biological replicates. (C) The Arabidopsis targets of Bc-sRNAs were suppressed after B. cinerea infection. PDF1.2, BIK1, and β-tubulin were used as controls. (D) The S. lycopersicum target gene MAPKKK4 was suppressed upon B. cinerea infection. Expression [(C) and (D)] was measured by means of quantitative RT-PCR by using actin as an internal control. Error bars indicate SD of three technical replicates. Similar results were seen in three biological replicates. (E) Coexpression of Bc-siR3.2 or Bc-siR5 with their host targets (HA-tagged) in N. benthamiana revealed target silencing by means of Western blot analysis. Coexpression of AtmiR395 or target site–mutated versions of target genes was used as controls. (F) Expression of YFP-MPK2 or its synonymously mutated version (YFP-MPK2-m) after infection of B. cinerea was observed with confocal microscopy. Coexpression of YFP-MPK2 and Bc-siR3.2 was used as a control. (G) Expression of the YFP sensors carrying a Bc-siR3.2 target site of MPK2 or a Bc-siR3.2 target site-m was analyzed after infection of B. cinerea. Samples were examined at 24 hours after inoculation. (Top) YFP. (Bottom) YFP/bright field overlay. Scale bars [(F) and (G)], 37.5 μm. Error bars indicate SD of 20 images [(F) and (G)]. The asterisk indicates significant difference (two-tail t-test; P < 0.01). Similar results were obtained in three biological replicates in (E) to (G).

Fig. 2. Bc-sRNAs trigger silencing of host targets that are involved in host immunity

(A) Expression of Bc-siR3.1, BcsiR3.2, or Bc-siR5 in transgenic Arabidopsis ectopically expressing Bc-sRNAs under the Cauliflower Mosaic Virus promoter 35S (Bc-sRNAox) was examined by means of Northern blot analysis. Highly expressed lines were selected for the following experiments. (B) Bc-sRNAox lines showed constitutive silencing of respective Bc-sRNA target genes measured with quantitative RT-PCR. Two independent lines for each Bc-sRNA were examined. Similar results were observed in two generations of the selected transgenic lines. (C) Bc-sRNAox plants exhibited enhanced disease susceptibility to B. cinerea as compared with wild type. (D) Loss-of-function mutants of Bc-siR3.2 and Bc-siR5 targets mpk1 mpk2 and wak displayed enhanced disease susceptibility. In all pathogen assays [(C) and (D)], lesion sizes were measured at 96 hours after inoculation. Error bars indicate the SD of 20 leaves. (E) Biomass of B. cinerea was measured with quantitative PCR at 96 hours after inoculation. Error bars indicate SD of three technical replicates. For (C), (D), and (E), similar results were obtained from three biological repeats. (F) VIGS of MAPKKK4 exhibited enhanced disease susceptibility to B. cinerea in S. lycopersicum (examined at 72 hours after inoculation) as compared with control plants (TRV-RB). RB is a late-blight resistance gene that is not present in tomato. We chose to use a TRV vector with a fragment from a foreign gene as a control to eliminate the potential side effect of viral disease symptoms caused by TRV empty vector. Spray inoculation was used because silencing sectors are not uniform within the VIGS plants. Three sets of experiments with each of 6 to 10 plants for each construct were performed, and similar results were obtained. The asterisk indicates significant difference (two-tail t-test, P < 0.01) in (C) to (F).

Fig. 3. Bc-sRNAs hijack Arabidopsis AGO1 to suppress host immunity genes

(A) Loading of Bc-siR3.1, Bc-siR3.2, and Bc-siR5 into Arabidopsis AGO1 during infection was detected with AGO1-IP followed by RT-PCR. AGO1 from B. cinerea–infected leaves harvested at 24, 32, and 48 hours after inoculation was pulled down by AGO1 peptide antibody, and RNA was extracted from the AGO1-IP fraction. As a control, noninfected leaves mixed with B. cinerea mycelium (at least twice as much as that in B. cinerea–infected leaves at 48 hours after inoculation) were used to rule out any binding between AGO1 and Bc-sRNAs during the experimental procedures. Similar results were obtained from at least three biological repeats. (B) Arabidopsis ago1-27 exhibited reduced disease susceptibility to B. cinerea as compared with the wild type. Lesion size of at least 20 leaves and fungal biomass were measured at 96 hours after inoculation. (C) Silencing of MPK2, MPK1, PRXIIF, and WAK during B. cinerea infection was abolished in ago1-27. (D) Arabidopsis dcl1-7 exhibited enhanced disease susceptibility to B. cinerea as compared with the wild type. Similar results were obtained from three biological repeats [(B) to (D)]. The asterisk indicates significant difference (two-tail t-test, P < 0.01) in (B) and (D).

Fig. 4. B. cinerea dcl1 dcl2 double mutant is compromised in virulence

(A) B. cinerea dcl1 dcl2 double mutant, but not dcl1 or dcl2 single mutants, was impaired in generating Bc-siR3.1, Bc-siR3.2, and Bc-siR5 as revealed with RT-PCR. B. cinerea dcl1 dcl2 double mutant, but not dcl1 or dcl2 single mutants, produced much weaker disease symptoms than did the wild type in (B) Arabidopsis and (C) S. lycopersicum, as demonstrated by the lesion size measured of 20 leaves at 96 and 48 hours after inoculation, respectively. Similar results were obtained from three biological repeats. (D) Expression of the sensor YFP-Bc-siR3.2 target site was silenced by wild-type B. cinerea upon infection, but not by the dcl1 dcl2 mutant at 24 hours after inoculation. Scale bar, 75 μm. Error bars indicate SD of 20 images. Experiments were repeated two times with similar results. (E) B. cinerea dcl1 dcl2 mutant was compromised in suppression of MPK2, MPK1, and PRXIIF in Arabidopsis and MAPKKK4 in S. lycopersicum. Similar results were seen in two biological repeats. (F) Arabidopsis Bc-siR3.1ox and Bc-siR3.2ox lines were more susceptible to B. cinerea dcl1 dcl2 strain than was Col-0 wild type. (G) Enhanced disease phenotype of dcl1 dcl2 infection was also observed on TRV-MAPKKK4–silenced S. lycopersicum plants. Experiments in (F) and (G) were repeated three times with similar results. B. cinerea biomass was quantified at 96 hours after inoculation. The asterisk [in (B), (C), (D), (F), and (G)] indicates significant difference (two-tail t-test; P < 0.01).