Project Title: Investigating RNA silencing as a method of controlling Fusarium verticillioides infections Principal Investigator: ???? Project Summary Fusarium verticillioides is a fungal plant pathogen that causes widespread diseases of corn including ear and stalk rots. This fungus is also a prolific producer of the mycotoxin fumonisin B1 which can cause serious diseases in animals and humans that consume contaminated grain. RNA interference is a mechanism that is conserved among many eukaryotes that is thought to control viral infections. In the last few years ways to use RNA silencing as a tool for research and as a possible means of controlling plant pathogens have been investigated. The use of double stranded RNA sequences has been shown to induce gene silencing in vitro and in planta. The prospects of using RNA silencing to control fungal infections has been investigated for several plant pathogens. In this proposed study, mechanisms of how double stranded RNA molecules get into fungal cells will be investigated using gene expression studies and restriction enzyme mediated integration to generate fungal mutants that are unable to undergo RNA silencing. RNA silencing that would be effective against this fungus in plants would be a major accomplishment as this pathogen has proven very difficult to control. By determining which genes from Fusarium verticillioides will allow for effective control of the pathogen with RNA silencing all that will remain to be seen is if this will work in planta. Additionally, determining how double stranded RNA is taken into host cells may have larger implications for many organisms as RNA silencing systems are highly conserved and would likely lead to discoveries in other organisms. Introduction The filamentous fungus Fusarium verticillioides causes serious losses in corn as a result of root, ear and stalk rot and seedling blight. Fusarium verticillioides also can cause symptomless, systemic infections in corn which are difficult to detect. This pathogen is found globally and is thought to be one of the most common fungal infections of corn (Oren, Ezrati et al. 2003). This pathogen can be found infecting crops in every corn growing region on earth. Large amounts of time and money have gone into finding effective methods of controlling the diseases caused by this pathogen. However, the application of expensive fungicides is the only method currently employed by growers and without complete success. Breeding for resistance has been successful to some extent but the unpredictable nature of the fungus and the effects of environmental conditions on the severity of infection make these resistant varieties highly variable in their effectiveness when grown in different climates. Resistant varieties that look good in greenhouse studies often do not perform to expectations in larger field trials. Additionally, breeders usually select for an observable reduction in disease symptoms but this does not cover the systemic infections that often occur. One of the major concerns when this fungus infects corn is the secondary metabolites that it produces, mainly fumonisin B1 (FB1). To date there are 28 fumonisins that have been identified that Fusarium verticillioides produces, however FB1 is the most abundant and the most toxic (Williams, Glenn et al. 2007). These mycotoxins are found even in systemically infected, asymptomatic tissues making the testing of grain necessary even in the absence of ear rot (Desjardins and Proctor 2007). Fumonisin B1 inhibits sphingolipid metabolism which makes it harmful to humans and animals when consumed in contaminated grain. In horses, FB1 causes leukoencephalomalacia that can be fatal in just a few days. In pigs, pulmonary edema is the main result of consuming contaminated grain while in humans neural tube defects and esophageal cancer have been linked (Desjardins and Proctor 2007). This has led the United States and the European Union to set strict limits to the amount of fumonisins that can be present in corn based food and animal feed. In the past few years the phenomenon of RNA interference (RNAi) has been identified and become better understood. In many eukaryotes RNA interference is a mechanism used to deal with viral infections. The system functions by recognizing double stranded RNAs which are commonly found during viral infections. In plants, an RNase enzyme called Dicer recognizes double stands of RNA and cuts them into 19 to 24 base pair fragments (Bernstein, Denli et al. 2001). These small RNA fragments are then joined into the RISC complex of proteins which then scans mRNA in the host cell looking for homologous sequences. Once homologous sequences are identified they are degraded so that translation cannot occur. Since RNAi has become better understood the researchers have begun to use this as a tool for targeted gene silencing and also have begun to investigate using RNA silencing as a means to control plant pathogens. In addition to RNA silencing being investigated as a means to control plant pathogens, using RNA silencing to study fungal genes has proven beneficial. Traditional methods that have been used to knock out genes in fungi have had little success due to the low frequency of recombination and incomplete elimination of the target gene. Additionally, knock outs are not effective if more than one copy of a gene exists. Gene silencing can overcome this problem. In obligate biotophs gene knockouts have been even more difficult to obtain as it is impossible to grow some of these organisms in culture. Work has been done showing that plant pathogenic nematodes and certain insects that feed on plants have been shown to be reduced when they feed on transgenic plants expressing a hairpin RNA construct targeting critical genes for the pathogen (Huang, Allen et al. 2006; Mao, Cai et al. 2007). The hairpin structure is used to form the double stranded RNA necessary to induce gene silencing. Additionally, work has been done with the obligate biotroph Blumeria graminis showing that in transgenic wheat and barley that are expressing double stranded RNA (dsRNA) for critical fungal genes there was a reduction in fungal development on the plants (Nowara, Gray et al. 2010). Blumeria graminis produces haustoria which are specialized feeding structures that form a complex interaction between the plant and fungal plasma membranes where it is hypothesized that the exchange of small RNAs happens. It has been shown recently that transgenic tobacco plants could induce gene silencing in Fusarium verticillioides cells (Tinoco, Dias et al. 2010). Fusarium verticillioides does not produce haustoria so how the plant passes small RNAs to the fungus can only be speculated. F. verticillioides grows in the intercellular spaces of the plant where it can live as a biotroph only entering plant cells to reproduce, leaving the neighboring cells uninfected (Oren, Ezrati et al. 2003). I have been working with Fusarium verticillioides for several years and have gained a useful understanding of its interactions with corn. I have worked extensively in making fungal knockouts using traditional split marker recombination and the problems associated with trying to obtain fungal mutants. Additionally, I have utilized suppression subtraction hybridization in previous work and have a good working knowledge of the process. In order to gain fundamental knowledge about RNA silencing in Fusarium verticillioides I hypothesize that double stranded RNAs are actively taken up by the fungal cells and when exposed to double stranded RNAs for essential fungal genes growth will be severely limited. To accomplish the objectives of this application, I will pursue two specific aims: I.
Identify how siRNAs enter cells of Fusarium verticillioides II.
Identify target genes that are effective in controlling the growth of this fungus. My expectations are that, at the conclusion of the proposed period of support, I will have determined 1) whether or not uptake of miRNAs into fungal cells is an active process that requires changes in gene expression. 2) I will have gained insight into the way fungal cells uptake small RNAs from plant cells without haustoria. This work will have implications for other filamentous fungi since a majority of the work done on molecular interactions between plant pathogenic fungi and their hosts has been done in fungi that form haustoria. 3) I will have identified which genes would be the most useful if used in transgenic plants to control Fusarium verticillioides infection. Determining which genes will be most effective in limiting F. verticillioides infection in planta will be the starting point for developing transgenic crops that can induce silencing of key fungal genes resulting in limited infection. Rationale and Significance As previously mentioned, successful work has been done in different fungal plant pathogen systems that have resulted in an effective means of control using transgenic plants expressing double stranded RNAs for essential pathogen genes. To date the only work done on gene silencing with Fusarium verticillioides has been a study showing that dsRNA could transfer from transgenic tobacco into the fungus and effectively silence the target gene. Understanding how these dsRNAs are transferred from the host to the pathogen will shed light on an area of RNA silencing that will have implications for other organisms as well since RNA silencing seems to be highly conserved. More work needs to be done to understand the specific components in Fusarium verticillioides since there has been some variation in the RNA silencing systems of several filamentous fungi. Utilizing gene silencing as a means of control for Fusarium verticillioides could give growers added protection against this hard to control fungus. Also, this technology could be utilized in areas where expensive fungicides are not available and high levels of fumonisins in grain lead to serious health problems. In order for this to work a basic understanding of how the fungus is interacting with the plant is essential. Understanding which genes will make the most effective candidates for complete control of the fungus will be important in the future. It has been shown that fungi that have had genes silenced by RNAi maintain the silencing though many generations so fungi who are not fatally silenced will be able to be maintained in culture collections that could benefit other areas of Fusarium research since traditional knockout experiments are not very effective. Research Design and Methods Specific Aim #1: Identify how siRNAs enter cells of Fusarium verticillioides Introduction. It has been shown that plants expressing a double stranded RNA sequence can pass those RNAs to fungal cells where they can cause silencing in the fungal cells. However, to date no studies have been able to prove exactly what is happening to transfer the RNA from the host to the pathogen. Is it merely as passive process that allows the small RNAs to enter the fungal cells along with all the nutrients that the fungus is taking in from the plant cells? Or are there specific receptors and processes that take these RNAs into the cells to starts the process of gene silencing? The system of RNA silencing is hypothesized to be a means for dealing with viruses with RNA genomes. During viral replications double stranded RNA is present in infected cells and triggers the host response by silencing those genes to limit viral infection. This does not explain if plants have a mechanism for the uptake of RNA from outside the cell. However it has been shown in mammals that silencing can be spread from cell to cell (Bernstein, Denli et al. 2001). Gene silencing in nematodes can be induced by adding dsRNAs to food media along with a fluorescence tag (Urwin, Lilley et al. 2002). It has been shown that the fluorescence tag enters the gut of the nematode and after feeding gene silencing is induced implying that the dsRNAs are taken into the cells of the nematode where they are joined to the RISC complex for silencing. However, no studies have shown how the RNA gets into the cells of the nematode. Recently, total internal reflection fluorescence microscopy was used to observe single microRNAs (miRNA) that has been fluorescently labeled (Chan, Chan et al. 2010). While this technique seems promising it still has some roadblocks including determining if the fluorescently labeled RNA is able to pass into the cells. Also, there are several organelles in plant cells that auto fluoresce making searching for these very small fluorescent RNAs difficult under fluorescence microscopy. The ultimate goal would be to tag and observe a single double stranded RNA moving from the host cell to the pathogen cell but currently there are no methods that allow this to be done. Instead, other evidence must be gathered to support the movement of these RNAs. It is our hypothesis that there are changes in gene expression and active processes occurring in the plant that are activated in the presence of double stranded RNA outside of the cell. Experimental Design. Evaluate changes in gene expression in response to siRNAs. To test whether there are changes in gene expression in the fungus as a result of exposure to small double stranded RNAs, a suppression subtraction hybridization (SSH) will be used. Although a microarray analysis may seem like the more effective method to screening for gene expression, in this case there is no microarray currently available for Fusarium verticillioides. This technique also does not have the limit of only observing genes present on a chip, SSH will get results for things that may not have been seen before making it appealing for this research. To test whether there are changes in gene expression in response to small RNAs cultures of Fusarium verticillioides strain M‐3125 that has been modified to express green fluorescent protein (GFP) will be grown from single spores on potato dextrose agar. After colonies have germinated, single colonies will be transferred to individual agar plates so that only one strain of fungus is growing on each plate. RNAs will be generated by reverse transcriptase PCR so that many copies of the GFP RNA can be made. In order to determine which time points may be of the most interest a solution containing double stranded RNA for the green fluorescent protein will be added to the colonies and checked at intervals under a fluorescence microscope to observe and quantify changes in the amount of fluorescence. A measureable decrease in fluorescence will indicate that the fungal cells have taken the RNA in and the GFP is being silenced. Once the time point where this begins in determined points prior to that time will be used to study changes in the gene expression. To obtain samples for suppression subtraction hybridization colonies will be grown of single spores as was done for the time point study. Once single colonies are grown to approximately 1 inch in diameter they will be treated with a solution of 1X TE buffer containing double stranded RNA for the green fluorescent protein. Control colonies will be treated only with 1X TE buffer that does not contain RNA. Samples from four time intervals prior to the determined time point will be sampled for SSH with the RNA exposed colonies being the tester and the control colonies being the driver. Total RNA will be extracted from each colony using Trizol. The total DNA from 10 treated colonies and 10 control colonies will be pooled to eliminate variation within each treatment. A commercially available kit for suppression subtraction hybridization will be used to compare the two treatments. After the SSH protocol is completed the resulting DNAs will be sequenced to determine their identity. The genome for Fusarium verticillioides has been sequenced so determining the identity of obtained genes can be done using bioinformatics software. Screen Fusarium verticillioides mutants for the absence of RNA induced gene silencing. In addition to the gene expression study, testing mutants for the inability to undergo RNA induced silencing will further expand our understanding of how RNA silencing works in filamentous fungi. In order to determine if it is possible to have mutant Fusarium verticillioides that are unable to take in dsRNA mutants must be generated. This technique will also likely result in the mutation of integral silencing genes such as the RISC complex of proteins and RNA dependent RNA polymerases. This is also of interest since the genes involved in silencing in Fusarium verticillioides have not been characterized. In several other filamentous fungi interesting observations have been made when genes integral to RNA silencing have been mutated. In Neurospora and Aspergillus nidulans RNA dependent RNA polymerases are not required for the generation of siRNAs and in filamentous fungi there have been several other unique differences in the RNA silencing pathway that have been discovered (Li, Chang et al. 2010). To generate Fusarium verticillioides mutants, restriction enzyme‐mediated integration (REMI) will be used. Fungal protoplasts will be generated so that plasmids can be inserted that contain the hygromycin resistance marker gene. This will allow for the transformed fungal cells to be separated from non‐transformed cells by growing them on media containing hygromycin. Details of the fungal transformation protocol and plasmid generation have been described by Seong et al 2005. After the mutants are generated they will be screened for the lack of RNA silencing. In order to determine which colonies are unable to uptake dsRNA, the colonies will be grown on PDA plates with one mutant per plate. The fungi will then be treated with a solution containing double stranded RNA that if taken into the fungi will cause a fatal silencing of essential genes and allowed to soak for a period of time. Any colonies that live will be assumed to be unable to take up the RNA. To confirm that the mutants do not have the RNA inside them the colonies will be scraped off the plates and washed thoroughly to ensure all external RNA is removed. The total RNA will be isolated from each colony using Trizol. The RNA extracts will be screened for small RNAs corresponding to the RNA that was used to treat each colony. Once it is determined that there mutant colonies do not have the RNAs in them the mutants will be characterized to determine what the mutations are. Overall expectations for Specific Aim #1. By determining the changes in gene expression in response to miRNAs there are opportunities to shed light on several aspects of this system. First, it has never been shown specifically in Fusarium verticillioides how RNA induced gene silencing works. Clearly, since it has been shown that it does happen for this fungus the machinery involved must be present. There are some fungi such as Ustilago maydis that do not have the genes necessary to perform RNA induced gene silencing (Li, Chang et al. 2010). Understanding what genes are involved in Fusarium verticillioides will add to the knowledge about the widespread distribution and evolution of gene silencing in fungi. This aspect of the study will show which genes are regulating this interaction, including the expression patterns of the different components of the RNA silencing enzymes and proteins. Secondly, this study will start to reveal how small RNAs pass form a plant cell into a fungal cell. Even a negative result in this situation could be useful in indicating that this may be a passive process in which the RNA passes into the fungus with other nutrients that are being absorbed. It is likely that with the seemingly conserved nature of RNA silencing that uncovering the mechanisms involved in RNA trafficking could have widespread importance for other organisms. Specific Aim #2: Identify target genes that are effective at controlling growth of this fungus. Introduction. In order for RNA silencing to be useful as a tool to control Fusarium verticillioides infections in crops, silencing targets that greatly or totally reduce the growth of the fungus must be identified. These genes must also not have homology with plant genes as this would results in silencing in the plant and could cause problems in the plant. In Sclerotinia sclerotiorum type 2A phosphoprotein phosphatase has been shown by silencing to be essential for plant virulence and development (Erental, Harel et al. 2007). There have been very few genes that have been identified to specifically function as virulence factors in Fusarium verticillioides. As with resistance genes in plants, virulence genes in the fungi can be lost or evolve to escape selection since they are usually not essential for the function of the pathogen. This makes them less desirable as targets of silencing than genes that are essential for the life of the fungus such as metabolic pathways, housekeeping genes and structural components like chitin. These targets are predicted to be much more effective at controlling the pathogen via silencing. For some species of fungi whose mycotoxins are known to essential for causing disease targeting these mycotoxin genes would be an effective means of controlling the pathogen (McDonald, Brown et al. 2005). Unfortunately, the link between infection by F. verticillioides and fumonisin production is not as clear cut. Many studies have been conducted to show the link between fumonisin production and disease symptoms but the results have been mixed and a clear link has not been shown as of yet. As was done by Nowara et al. 2010 a study of the genes that are turned on during infection with a fungal pathogen give a good starting point to identify important genes that may have a significant impact if silenced in the pathogen. Unfortunately, in the case of Fusarium verticillioides no transcriptomics experiments have been done to identify genes that are differentially expressed during infection so this will need to be completed to identify these targets for this fungus. It is our hypothesis that identifying important genes for Fusarium verticillioides by a bioinformatics study of the genome sequence and by using suppression subtraction hybridization that we will be able to select effective targets for control of this fungi through silencing. Experimental Design. In order to identify target genes of interest the genome sequence of Fusarium verticillioides will be used to identify genes involved in metabolism and other essential functions in the cell. Additionally, to determine genes that are involved in infection, suppression subtraction hybridization (SSH) experiment will be completed to compare the expression of genes that are turned on when the fungi is infecting plant material. To do this, colonies of the fungus will be grown on PDA media. In order to activate genes that would be activated during infection the fungus will be transferred to media containing ground and sterilized corn. While this may not be an identical environment to being inside a plant, the ability to recover the fungi from an agar plate makes this approach an appealing one. If RNA was extracted from an infected corn plant all of the corn genes used for half of the subtraction would wind up in the final product. While it would be possible to use the genome sequences of these two organisms to determine which genes belong to which organism, it is likely that the plant genes would be highly represented in the SSH and may obscure important results that would be obtained otherwise. When a microarray chip for this fungus has been created, this experiment will benefit from being redone to get a more complete look at the genes that are differentially regulated during infection. Once the sequences are identified primers will be designed and so that the genes of interest can be amplified from cDNA copies of the extracted RNA making approximately 40 base pair segments that contain a coding region for the gene. These fragments will be used as templates to create sense and antisense copies of the genes using a commercial RNAi kit. Wild type Fusarium verticillioides strain M‐3125 will be grown from single spores on PDA media. Once colonies are a few millimeters in size they will be removed and transferred onto individual plates so that only one colony is growing per plate. Once these colonies are ½ inch in diameter they will be treated with the generated RNA fragments. Each fragment will be evaluated on 10 colonies so the results can be combined. To measure the effect that these RNAs have on the fungal colonies they will be incubated with the RNA fragments for 48 hours and then the diameter of the colonies will be re‐measured. The changes in each colony size will be averaged between each replicate for the treatment. Control colonies will be grown alongside the treated colonies for comparison. The change in colony size as compared to the wild type will be measured. Overall expectations for Specific Aim #2. For the suppression subtraction hybridization of Fusarium verticillioides on regular or corn based media, important results pertaining to this study and others will be obtained. To date, no work has shown what genes are differentially regulated when this fungi is infecting its host. The results of this study will advance our knowledge of the relationship between the fungus and its host and hopefully lead to better methods of control. After SSH is completed and other candidate genes are identified from the F. verticillioides genome, I expect that many of the colonies that are treated with essential genes will be reduced if not completely inhibited in their growth. This knowledge will be useful in advancing the use of gene silencing for control of fungal plant pathogens since it is likely that metabolism genes and structural components are fairly well conserved among groups of filamentous fungi. While society is currently hesitant to allow genetic modification of crops there may be less resistance to inserting small RNA molecules that do not comprise and entire gene or code for a full protein. Future Directions. There are many studies that will need to be done to follow up on the work discussed here since RNA silencing is a relatively new technology. It remains to be seen how stable silencing could be in crops or whether or not it would be an effective means for control when used on a large scale. It has also not been investigated if there is a threshold amount of infection that could overcome gene silencing. The work accomplished with this proposal will start the work that needs to be done to move towards the goal of effectively utilizing RNA silencing. There is also work that could be done to determine if RNA silencing could be useful in controlling mycotoxins contamination. In the case of Fusarium verticillioides a gene for silencing fumonisin production would be unlikely effective at controlling the fungus since the production of fumonisin has not been shown to be associated with virulence. There may be some benefit for increased grain quality but there would still be damage caused by ear and stalk rots. Information gained by studying genes that are differentially expressed during dsRNA exposure will need follow up studies to more closely examine what these genes are doing and how they are bring regulated. Timeline In the first year of this study the timing study for gene silencing will be started and completed. After this is done the gene expression study will started for the experiment relating to dsRNA uptake. The gene expression study will be completed in the next year. Following the gene expression study, generation of Fusarium verticillioides mutants will be generated. Screening the mutants will begin in the third year of this study and will likely take at least one year. During years three and four the bioinformatics study and SSH experiment to identify candidate genes will be completed. During year four RNA silencing of Fusarium verticillioides with identified candidate genes will be concluded. Works Cited Bernstein, E., A. M. Denli, et al. (2001). "The rest is silence." RNA 7: 1509‐1521. Chan, H.‐M., L.‐S. Chan, et al. (2010). "Direct quantification of single‐molecules of microRNA by total internal reflection fluorescence microscopy." Analytical Chemistry 82(16): 6911‐6918. Desjardins, A. E. and R. H. Proctor (2007). "Molecular biology of Fusarium mycotoxins." International Journal of Food Microbiology 119: 47‐50. Erental, A., A. Harel, et al. (2007). "Type 2A Phosphoprotein Phosphatase Is Required for Asexual Development and Pathogenesis of Sclerotinia sclerotiorum." Molecular Plant‐Microbe Interactions 20(8): 944‐954. Huang, G., R. Allen, et al. (2006). "Engineering broad root‐knot resistance in transgenic plants by RNAi silencing of a conserved and essential root‐knot nematode parasitism gene." Proceedings of the National Academy of Sciences 103(39): 14302‐14306. Li, L. D., S. S. Chang, et al. (2010). "RNA interference pathways in filamentous fungi." Cellular and Molecular Life Sciences 67(22): 3849‐3863. Mao, Y.‐B., W.‐J. Cai, et al. (2007). "Silencing a cotton bollworm P450 monooxygenase gene by plant‐
mediated RNAi impairs larval tolerance of gossypol." Nature Biotechnology 25(11): 1307‐1313. McDonald, T., D. Brown, et al. (2005). "RNA silencing of mycotoxin production in Aspergillus and Fusarium species " Molecular Plant‐Microbe Interactions 18(6): 539‐545. Nowara, D., A. Gray, et al. (2010). "HIGS: Host‐induced gene silencing in the obligate biotrophic fungal pathogen Blumeria graminis." The Plant Cell 22: 3130‐3141. Oren, L., S. Ezrati, et al. (2003). "Early Events in the Fusarium verticillioides‐Maize Interaction Characterized by Using a Green Fluorescent Protein‐Expressing Transgenic Isolate." Applied and Environmental Microbiology 69(3): 1695‐1701. Tinoco, M. L. O., B. B. Dias, et al. (2010). "In vivo trans‐specific gene silcening in fungal cells by in planta expression of a double‐stranded RNA." BMC Biology 8(27). Urwin, P. E., C. J. Lilley, et al. (2002). "Ingestion of double‐stranded RNA by preparasitic juvenile cyst nematodes leads to RNA interference." Molecular Plant‐Microbe Interactions 15(8): 747‐752. Williams, L. D., A. E. Glenn, et al. (2007). "Fumonisin disruption of ceramide biosynthesis in maize roots and the effects on plant development and Fusarium verticillioides‐induced seedling disease." Journal of Agricultural and Food Chemistry 55: 2937‐2946. Budget Cost Budget Item Personel‐Postdoc and 1 Graduate student Graduate Student Tuition Travel Materials and supplies (detailed below) *PCR supplies and enzymes *Suppression subtraction hybridization kits *Primers *Sequencing *Vectors *Consumables ‐ tips, tubes, plates, etc Year 1 $49,242.00
$1,980.00
$1,000.00
$30,000.00
$3,000.00
$10,000.00
$1,000.00
$10,000.00
$3,000.00
$3,000.00
Year 2 $50,719.00
$2,063.00
$1,030.00
$30,900.00
$3,000.00
$10,000.00
$1,000.00
$10,000.00
$3,000.00
$3,000.00
Year 3 $52,241.00 $2,150.00 $1,061.00 $31,827.00 $3,000.00 $10,000.00 $1,000.00 $10,000.00 $3,000.00 $3,000.00 Year 4 Total $53,808.00 $206,010.00
$2,240.00
$8,433.00
$1,093.00
$4,184.00
$32,782.00 $125,509.00
$3,000.00 $12,000.00
$10,000.00 $40,000.00
$1,000.00
$4,000.00
$10,000.00 $40,000.00
$3,000.00 $12,000.00
$3,000.00 $12,000.00
$464,136.00