Great talk about Cross-kingdom RNAi by Hailing Jin

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Great talk about Cross-kingdom RNAi by Hailing Jin

Last week we were treated by a fantastic seminar from Hailing Jin from the University of California. In the over-airconditioned seminar room we quickly forgot about the cold while she introduced us in the world of cross-kingdom RNAi and small RNA trafficking between plants and fungal pathogens. She started with telling us how small RNAs are formed from their precursors using Dicer-like (DCL) proteins and how these small RNAs are incorporated into AGO proteins to induce gene silencing, which we call RNA interference (RNAi). And how RNAi is a conserved regulatory mechanism across almost all eukaryotic cellular processes. We then were introduced to her pathogen of choice in her study of plant-fungal pathogen interaction, Botrytis cinerea (Bc). Bc is actually around us in the air and is probably the one you see when your fruit of veg has gone mouldy. However, the main reason she used them is that the have their own small RNA machinery and can thus process small RNAs by themselves. Part of the way how Bc infects plants is that after penetrating the plant cell, Bc secretes small RNAs into the plant cell. These Bc-small RNAs are then loaded by the plant onto its own AGO machinery, which then will silence the genes targeted by the Bc-small RNA. The result of this is an inhibition of the plant immune response, enhancing the virulence of Bc. Subsequent research by her and other groups has shown that similar mechanisms are used by fungi infecting mammalian cells, as well as by parasitic plants and bacteria and their interaction with their host.

Hailing Jin told us then the realisation that if small RNAs can travel from fungi to plant, they supposedly should be able to travel into the other direction as well. The trouble with this was that in order to be able to show that an organism has received small RNAs from another organism you need to be able to separate the two organisms from each other, before the isolation of RNA. As both plants and fungi have cell walls, which actually consist of different compositions, the separation should be possible using sequential protoplast purification. So she convinced a postdoc to give it a try or actually “Give it three tries, as when you try something new you might fail the first time, but with troubleshooting you will succeed the second or third time.” Therefore, with some tries a postdoc from her lab was able to do this by isolating Bc-protoplast from infected leaves. Subsequent analysis of the small RNAs of these Bc-protoplasts showed that not only they contained small RNAs originating from plants, but also that these small RNAs where actively transported to the fungal cells.

In the following quest to find out how these plant-small RNAs were delivered to the fungi Hailing Jin looked into the possible delivery options that are known. One of these are extracellular vesicles, which have been implicated in the transport of small RNAs in mammalian cells. In order to find out if in plants these extracellular vesicles contained small RNAs they collected the extracellular fluid from the phloem from both control and Bc infected leaves. This showed not only that these extracellular vesicles contained small RNAs, but also that when coming from Bc-infected leaves that these small RNAs matched those found in Bc-protoplast from infected leaves. They then went on to confirm that these extracellular vesicles are exosomes, by showing that the plant exosome markers TETRASPANIN (TET), TET8 and TET9 are induced by Bc infection. Using TET8-GFP, they show that TET8-GFP accumulates at the infection site, were TET8-GFP labelled vesicles are secreted. Subsequent analysis of the uptake of these vesicles showed that they are readily taken up by Bc. To confirm that TET8 and TET9 are required for the vesicles transporting the small RNAs they created plants with a reduced expression of TET8 and TET9, and infected these plants with Bc. Not only were these plants now more susceptible to Bc infection, they also had a reduced transfer of plant-small RNAs to Bc. Indicating that the small RNAs that the plant transfers to Bc contribute to its defence against Bc. They then looked into which genes the plant-small RNAs targets in Bc. It turned out that a lot of these genes were actually involved in vesicle trafficking.

Haining Jin went then on to discuss that small RNA trafficking is a level of communication across-species, and how we can use this for plant protection from fungal pathogens. As this is an interesting topic that deserves a post on its own I will talk about that in more detail later. But I will finish with giving you the links to  Haining Jin’s articles including those discussing how cross-kingdom RNAi can be used for plant protection.

References

Weiberg et al., Science, 342:118-23, 2013
Cai et al., Science 360:1126-29, 2018
Weiberg et al., Current Opinion in Biotechnology, 32:207-215 2015
Huang et al., Cell Host and Microbe, 26: 173-182, 2019
Wang et al., Nature Plants 2:16151, 2016