Peter M. Waterhouse, Michael W. Graham and Ming-Bo Wang Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA Peter M. Waterhouse, Michael W. Graham and Ming-Bo Wang Presented by: Fang Gao Kehui Lian

History of RNAi in plants Co-suppression As we have learned in the previous lectures, RNA interference is a mechanism for gene silencing. The current model of RNAi started with the discovery of the effect of co-suppression of genes in transgenic petunia plants in 1990. It was observed that the introduced transgenes are sometimes not expressed, in other words, they were silenced. At the same time, the transgenes can also cause the silencing of the homologous endogenous plant genes. Stam, Mol and Kooter (1997)

Viral protein mediated resistance After this discovery, the mechanism of virus resistance and gene silencing in plants became the research interest of many scientists. Some studies showed that transgenic plants expressing viral proteins allowed plants to develop resistance against viral pathogens. The concept behind these studies was that viral proteins could function in regulating different stages of the life cycle of the virus, and that the transgenic plants expressing the viral proteins would disrupt this life cycle, thus allowing plants to obtain resistance. However, in some studies, it was found that some of the transgenic plants that expressed a nontranslatable, sense-stranded mRNA for the TEV CP gene were also immune to the virus.

Q: Some transgenic plants that expressed a nontranslatable, sense-stranded mRNA for the TEV coat protein gene were also immune to the virus WHY? Q: What does this result mean? Ans: The RNA sequence of the TEV CP gene, and not the coat protein itself can allow plants to develop immunity against the virus. Ans: The RNA sequence of the TEV CP gene, and not the coat protein itself can allow plants to develop immunity against the virus.

Post-transcriptional gene silencing PTGS Sequence-specific RNA degradation mechanism  Involves double-stranded RNA A mechanism that plants have evolved for protection from virus infection Also involved in protecting plant from transposons and any RNA that is expressed at a very high level Post-transcriptional gene silencing (PTGS) in plants is an RNA-degradation mechanism.

Later, to study the mechanism of this sequence specific RNA degradation or gene silencing process, researchers transformed virus-derived sense or antisense RNA into plants to see the effect on virus resistance.

Sense vs Antisense RNA Sense = Positive sense - Coding strand Antisense = Negative sense - Complementary to the coding strand - Non-coding Eg. 5´   C A U G   3´     mRNA 3´   G U A C   5´     Antisense RNA Sense vs Antisense The strand names actually depend on which direction you are writing the sequence that contains the information for proteins (the "sense" information), not on which strand is on the top or bottom (that is arbitrary).

Early RNA silencing model In the early 90s, it was proposed that gene silencing could be achieved by the base pairing of the sense RNA transcript of the transgene and the negative strand of the viral RNA, or small fragments of antisense RNA could be produced by an RdRp encoded in the host genome by using the transgene RNA as a template, and they would have the potential to base pair with both the transgenic and viral RNAs (Figure 1A). Lindbo et al. (1993)

RNA silencing model Lindbo et al. (1993) (i) and (ii) Antisense RNA binds to transgene (i) and/or viral (ii) RNA, blocking translation and thereby destabilizing the transcript. If the encoded protein is required for virus replication, the accumulation of viral genomic RNA is inhibited; (iii) and (iv), binding of antisense RNA fragments to transgene (iii) and viral (iv) RNAs recruits a double-strand (ds) specific RNase, which degrades the target transcript; and (v), antisense RNA binding masks a c/'s-acting element in the viral genome, preventing its interaction with a virus-encoded protein (VP). The VP-RNA interaction may be necessary for virus replication, movement, or other essential phases of the infection cycle. The inhibition of this interaction could therefore prevent virus accumulation in the inoculated plant. Lindbo et al. (1993)

Materials and Methods Potato virus Y (PVY) Protease gene (Pro) Tobacco transformation

Potato Virus Y (PVY) Positive sense strand RNA virus Positive-sense ssRNA viruses (Group IV) have their genome directly utilized as if it were mRNA, with host ribosomes translating it into a single protein that is modified by host and viral proteins to form the various proteins needed for replication. One of these includes RNA-dependent RNA polymerase (RNA replicase), which copies the viral RNA to form a double-stranded replicative form. In turn this directs the formation of new virions. Positive sense strand RNA virus Family: Potyviridae Genus: Potyvirus Symptom: potato tuber necrotic ringspot

Protease (Pro) gene When viral RNA is translated into a polypeptide sequence, that sequence is assembled in a long chain that includes several individual proteins (reverse transcriptase, protease, integrase). Before these enzymes become functional, they must be cut from the longer polypeptide chain. Viral protease cuts the long chain into its individual enzyme components which then facilitate the production of new viruses.

Hypothesis Test whether the introduction of gene constructs that produced mRNA transcripts capable of forming a duplex would be more or less effective at generating PTGS than constructs producing either sense or antisense mRNA alone. Q: What would be their expectations? Hypothesis Main objective: Test whether the introduction of gene constructs that produced mRNA transcripts capable of forming a duplex would be more or less effective at generating PTGS than constructs producing either sense or antisense mRNA alone. Q: What would be their expectations? If the dsRNA acts as a trigger for gene silencing, transformation of plants with the construct that expresses both sense and antisense forms of a gene should silence the gene more effectively than expressing the gene in either single polarities.

Expression of Sense or Antisense alone in Plants 5/57 Pro[s] plant lines 1/54 Pro[a/s] plant lines 10/49 Pro [s]-stop plant lines Immune to the PVY

Q:What is this construct used to demonstrate? The immunity to PVY cannot be conferred by the PVY Pro protein.

Q? More copies of transgene were found in immune plants than those in susceptible plants.

Expression of both Sense and Antisense Pro Genes Q?

ELISA Enzyme-linked immunosorbent assay Substrate Enzyme linked secondary antibody Detecting antibody capture antibody Sample (1) Plate is coated with a capture antibody; (2) sample is added, and any antigen present binds to capture antibody; (3) detecting antibody is added, and binds to antigen; (4) enzyme-linked secondary antibody is added, and binds to detecting antibody; (5) substrate is added, and is converted by enzyme to detectable form

Phenotype Symptom Susceptible Obvious mottling of leaves of 5-10 days, high level of virus Resistant Small patches of mottling or chlorotic lesions after more than 14 days Immune No symptoms, no virus accumulation

Inheritance of immunity phenotype 35S Pro[s]35SPro[a/s] 35S Pro[s]S4Pro[a/s] 35S Pro[a/s] 35S Pro[s]

Q? What does this result suggest?

Inverted repeat configuration PTGS

Pro gene expression in Parental plants Northern Blot

GUS reporter system Test promoter GUS The GUS reporter system (GUS: β-glucuronidase) is a reporter gene system GUS gene encodes the enzyme β-glucuronidase (GUS). This enzyme can cleave the substrate, will result in the production of an insoluble blue color in those plant cells displaying GUS activity. We can use this to test the activity of a promoter (in terms of expression of a gene under that promoter) in different tissues. But in this study, they wanted to see if the GUS reporter gene can be silenced by using a single, self-complimentary mRNA that can form a panhandle structure. https://www.mpipz.mpg.de/somssich

Silencing of a Reporter Gene by Using a Single, Self-Complementary mRNA Transgenic rice tissue constitutively expressing GUS from a single transgene was supertransformed by using vectors that contained the bar gene conferring bialaphos resistance. The supertransformed tissue was maintained on bialaphos selection medium for 3 weeks and then analyzed for GUS activity (Fig. 6). (C) Constructs used to express DGUS mRNA in rice. The DGUS ORF has a 231-bp deletion to prevent production of active GUS protein. With the exception of Gus[iyr] (iyr, inverted repeat), which is promoterless, the constructs are controlled by the ubiquitin promoter. UbiDGus[s] and UbiDGus[ays] contain the DGUS ORFs in a sense and an antisense orientation, respectively. The 3’ region of the transcription unit of UbiDGus[i/r] and DGus[i/r] is complementary to the 5’ region of the DGUS transcript. The predicted hairpin structure of such an mRNA is shown at the bottom.

Q: Why was a promoterless “panhandle” construct used? - To show that PTGS requires transcription

Rice Supertransformation and GUS activity assay Calli constitutively expressing GUS (HygR) Incubate for 2 days with Agrobacteria with various binary vector constructs (BialaphosR) Grow calli on Bialaphos+ Hygromycin media for 4 weeks GUS activity assay

Silencing of a Reporter Gene by Using a Single, Self-Complementary mRNA Calli supertransformed with the binary vector containing the bar gene and promoter-terminator cassette without the DGUS gene gave no silencing of the endogenous GUS activity. Supertransformation with DGUS in a sense or antisense orientation showed some silencing of the endogenous GUS activity. However, supertransformation with a construct with the inverted repeats expressing the DGUS allowing its 3’ end to form a duplex with the 5’ end of the transcript, to give a ‘‘panhandle’’ structure, gave almost 90% silencing of the endogenous GUS activity. Supertransformation with the panhandle construct from which the promoter had been deleted resulted in very little silencing. This result suggests that mRNA with the panhandle structure is an effective trigger for inducing silencing and operates in a way similar to dsRNA formed from two independent molecules.