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New Study Shows Suppression of Plant Immunity by Fungal Small RNAs

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University of California, Riverside researchers this month reported the discovery that a fungal pathogen known to infect a wide variety of vegetable and fruit crops is capable of hijacking a plant's RNA interference pathway to suppress host immunity, thereby enhancing its virulence.

To date, small RNAs have been identified in various fungi and oomycetes, but data has been inconclusive as to whether they regulate host-pathogen interaction. To answer this question, the scientists looked to Botrytis cinerea, the fungus responsible for gray mold disease and a key research model given its broad host range.

According to their paper in Science, the investigators profiled sRNA libraries prepared from B. cinerea-infected Arabidopsis thaliana leaves, as well as infected tomato leaves and fruits, at various timepoints after inoculation.

A total of 832 sRNAs were present in both the Arabidopsis and tomato libraries. There were more reads in these libraries than in cultured B. cinerea control libraries, with sequences matching the B. cinerea genome, but not Arabidopsis or tomato genomes.

The team discovered that 73 of the B. cinerea sRNAs could target host genes in both of the plants studied "under stringent target prediction criteria," the scientists noted. "Among them, 52 were derived from six retrotransposon long terminal repeats loci in the B. cinerea genome, 13 were from intergenic regions of 10 loci, and eight were mapped to five protein-coding genes."

The team selected three fungal sRNAs for further examination, noting that they were among the most abundant that were 21 nucleotides in length and had potential plant targets deemed likely to be involved in immunity.

Following B. cinerea infection in Arabidopsis, transcript levels of several of the sRNAs' predicted targets were suppressed. Among them were mitogen activated protein kinase 2 and 1, or MPK2 and MPK1; the oxidative stress-related gene peroxiredoxin, or PRXIIF; and cell wall-associated kinase, or WAK.

Meanwhile, two plant defense marker genes, which do not contain target sites for the fungal sRNAs, were highly upregulated, leading the researchers to conclude that suppression of some but not all genes is a result of sequence-specific sRNA interaction and not cell death within infected lesions.

Notably, the sRNA that silenced MPK1 and MPK2 was also enriched in tomato plant leaves following B. cinerea infection, and inhibited expression of MAPKKK4, another member of the MAPK signaling cascade in the tomato plant.

The scientists then confirmed that the target suppression observed was triggered by the sRNAs using coexpression assays in Nicotiana benthamiana.

"Expression of hemagglutinin-epitope tagged MPK2, MPK1, and WAK was reduced when they were coexpressed with the corresponding [sRNAs] but not when coexpressed with Arabidopsis [microRNA] miR-395, which shared no sequence similarity," they wrote in Science. "The silencing was abolished, however, when the target genes carried a synonymously mutated version of the relevant" B. cinerea sRNA.

Suppression of tagged MPK2 was also observed after B. cinerea infection, but not when the sRNA's target site was mutated.

Next, the scientists evaluated the impact the B. cinerea sRNAs had on plant immunity, generating transgenic Arabidopsis plants that ectopically expressed the three sRNAs under investigation.

Although the plants showed normal morphology and development in the absence of pathogen challenge, even though the target genes were suppressed, all three displayed "enhanced susceptibility" to infection upon introduction of B. cinerea.

Meanwhile, tomato plants in which MAPKKK4 was suppressed using a virus-induced gene silencing approach showed enhanced disease susceptibility in response to B. cinerea and contained over 15 times more fungal biomass than control plants.

In order to confirm that the sRNAs suppressed their targets using their host's RNAi pathways, the investigators immunoprecipitated Argonaute 1 — the primary component of the RNA-induced silencing complex that enables RNAi — from Arabidopsis and identified the three sRNAs they have been studying in Ago1-associated fraction pulled from B. cinerea-infected plant samples but not controls.

Further, the scientists observed a reduced disease susceptibility in Arabidopsis plants in which Ago1 was mutated following B. cinerea inoculation versus wild-type plants, while a B. cinerea mutant that cannot produce the sRNAs under investigation showed reduced pathogenicity on both Arabidopsis and tomato.

Thus, the sRNAs "evidently hijacked host RNAi machinery by loading into Ago1," with the resulting complex suppressing host immunity genes, the researchers wrote.

"The implications of these findings may extend beyond plant gray mold disease caused by B. cinerea and suggest an extra mechanism underlying pathogenesis promoted by sophisticated pathogens with the capacity to generate and deliver small regulatory RNAs into hosts to suppress host immunity," they concluded.

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