I have not come across the use of siRNAs in fungi and I have thus no expertise to comment on your question; but it is possible to upregulate a gene's expression using oligonucleotide duplexes. It is easily explainable when the target sequence is upstream of the canonical TSS and when there is an anti-gene transcription occurring (for example, see Janowski, B.A. and D.R. Corey, Minireview: Switching on progesterone receptor expression with duplex RNA. Mol Endocrinol, 2010. 24(12): p. 2243-52.).

An alternate pathway to activate the gene is to use triplex-forming oligonucleotides (TFOs) that have successfully been demonstrated in yeast (see Ghosh, M.K., et al., Targeted activation of transcription in vivo through hairpin-triplex forming oligonucleotide in Saccharomyces cerevisiae. Mol Cell Biochem, 2005. 278(1-2): p. 147-55.). 

Does any of the above seem to explain your observation? I would be looking forward to learning what you find.

I use RNAi extensively in human cells and have come across this phenomenon on occasion. One possible explanation is that the siRNA does not lead to mRNA cleavage/degradation, but rather blocks translation of the protein much like miRNAs often do. If the protein goes down, it may trigger a feedback loop leading to increased transcription of the gene you are trying to knock down (this will depend on how the gene is normally regulated and whether feedback loops are involved), and increased transcript levels. I have also see increased protein levels on occasion, which could be again a kind of feedback (increased protein stability in response to mRNA loss) or an off-target effect (increased interferon signaling, stress signaling, etc.) which results in upregulation of the gene that is also compounded with the siRNA not working especially well on the target gene. My general rule of thumb is to use two or three siRNA that 1) knock down your protein well (mRNA is less important/informative), and 2) manifest similar phenotypes to one another to rule out an off-target phenotype. I hope this helps and good luck!

This paper shows that siRNAs can have some unpredictable off-target effects, including increases in gene expression: http://www.pnas.org/content/101/7/1892.long

Thanks everyone for the responses. There seems to be much we don't understand about these systems and the fungus i'm working with is probably much more of a black box.  Actually, my assay is indirect so I still need to confirm the higher expression with the addition of the siRNA. However, the pathway hinges on the gene i'm targeting to it makes sense.  

That paper from Francis Collins talks about activation of "off-target" genes; while the question here is activation of "target" genes. The explanation has to be different.

Dear Thomas,

in addition to all the important indications from the collegues above, you should check for alternative splicing variants. by inactivating a specific mRNA, your siRNA could induce the up-regulation of an alternative messenger from the same gene.

Many Thanks Amilcare, 

I'm not detecting any exons in my gene and haven't found a great prediction program for fungal splicing. However, I will keep your recommendation in mind as I progress through this. Also, the 2 siRNA that cause increases in gene expression bind at very different location on the mRNA.   

A while back (~2005) there was a paper in a high profile journal that described some microRNAs that induced gene expression and that this induction was dependent on he position of the cell cycle. 

We tried to down-regulate MYCN in Rhabdomyosarcoma along with PAX-3-FOXO1 fusion. After 48 hours, the MYCN expression has increased in several attempts. Although, MYCN amplification is one possible explanation, but the depletion of MYCN and the activation of feedback loop could be the possible reason.

Some of my questions are: 

Is your target gene free from splice variant? Is there any copy number variation or amplification? What is the length of your amplicon? Have you tried with different set of primers? Is your qPCR results correlates with your Westernblot? Have you tried to see any interferon induction (immune response detection assay)?

PS: My experience is only with animal cell lines not with plat/fungi. 

In prokaryotes there are small RNAs that positively regulate gene expression. I believe there are also examples for positive regulation by siRNA/miRNA in eukaryotes (sorry but I do not have a reference). Face it: introducing siRNAs into cells frequently works, but failures are rarely published. We are using a system that may somehow work in practice but we do not really know what happens in the cell.

Are you working with fungi cells? Then keep in mind fungi are not equal mammalian cells and also not equal bacterial cells. They are a step between and maybe the RISC and turn over rate is much faster.

When the gene is strongly regulated by a ste of transcription factors you see no knock-down because the activation is so fast that it is masked when you harvest your RNA.

I have knocked down a gene to 10-20% expression after 24 hours, after 48 hours the expression was 50-65% because 2 transcription factors were involved whereas another gene stayed at 10% for 24 and 48 hours respectively.

Hi Volker, 

Yes, I'm working with fungal mycelium.  I'm actually testing the gene the transcription factor is regulating.  In Fusarium graminearum,  Tri10 and Tri6 are transcription factors that regulate mycotoxin production, specifically Deoxynivalenol.  The Tri5 gene is the first essential step in the pathway.  I have a fungal strain that has a Tri5 promoter attached to GFP that I use as a biomarker for mycotoxin induction.  Currently, I'm testing through RT-PCR  to confirm that gene changes.  However, all of my measurement are indirectly based on the GFP fluorescence. 

It's very interesting that the transcription factors caused those results in your experiments.  Are you measuring expression with Delta-Delta CT? 

Would you like to see my poster? 

Cheers,

Tom

Hello Thomas,

I am using a Qiagen software REST to calculate the normalized ratio of my gene of interest to a reference gene.

In other question blogs I have read that the GFP is sometimes directly brought to the proteaosome when the cells do not accept the GFP protein very good. This will lead to a short half life time of the GFP in the cells. Some argued that a proteaosome inhibitor should be added in a control well and monitor the GFP fluorescence in hour steps.

I don´t know if this is a possible explanation for your fungal cells. Today I will ask a professor if this is the problem that some cell lines degrade the GFP very fast.  If it is true maybe it is also the same problem in your fungal cells.

I can have a look on the poster but I am not an expert for fungal gene expression ;-)

PS: I know that corn is tested when it is sold but I don´t know if these kits are sensitive enough for fungal cell culture.

Can you use one of thesse commercial fusarium detection kits to compare the levels of your transfected cells compared to your parental cells? This would circumvent the problem with your GFP for the first and you will know if it production is elevated or not. 

Tom,

We are also working on F.g. We have made a dcl2- mutant and Tri5 in this mutant is completely shut down while other Tri genes are significantly knocked down as well. Clearly siRNAs positively regulate the DON biosynthesis.

That is incredibly fascinating!

Dear Thomas,  Have you deal with this problem? I have met the same trouble as you have. 

Hi Thomas, I am working in silencing Blumeria graminis Fungal effectors and their targets in Hordeum vulagare. I am using Oligonucleotide application to down regulate  Malate dehydrogenase and Pathogenesis-related factor 5 activity. But resulting experiments show resisantce in hosts where susceptibility would be expected. I expect that activation by siRNA may be responsible for the over-expression. 

Hi Thomas

I am doing knockdown experiments on mammalian cells and two genes increase their expression instead of decreasing it. Actually, the effect is significant.

Zhanna Orlova did you resolved it? I am seeing an increased expression on mRNA levels.

Yes, I tried a ver low concentration of siRNA, the lowest possible and achieved knockdown !

Also a better primer design works, try to design towards 3'UTR of the CDS

Hi, can we knock down gene by upregulating the micro RNA which is targeting the particular gene? instead of using siRNA?

Nobody observed it using a plasmid carring a shRNA?

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