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Maize Defective Kernel Mutant Generated by Insertion of a Ds Element in a Gene Encoding a Highly Conserved TTI2 Cochaperone

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Maize Defective Kernel Mutant Generated by Insertion of a Ds Element in a Gene Encoding a Highly Conserved TTI2 Cochaperone Maize defective kernel mutant generated by insertion of a Ds element in a gene encoding a highly conserved TTI2 cochaperone Nelson Garciaa, Yubin Lia,1, Hugo K. Doonera, and Joachim Messinga,2 aWaksman Institute of Microbiology, Rutgers, The State University of New Jersey, Piscataway, NJ 08854 Contributed by Joachim Messing, April 5, 2017 (sent for review March 2, 2017; reviewed by L. Curtis Hannah and Clifford Weil) We have used the newly engineered transposable element Dsg to gene was cloned. Based on information from studies in animal tag a gene that gives rise to a defective kernel (dek) phenotype. and yeast systems, its gene product could be characterized as a Dsg requires the autonomous element Ac for transposition. Upon cochaperone hypothesized to enable early seed development excision, it leaves a short DNA footprint that can create in-frame by controlling the action of phosphatidylinositol 3-kinase-related and frameshift insertions in coding sequences. Therefore, we kinases (PIKKs) that regulate cellular signaling pathways related could create alleles of the tagged gene that confirmed causation to gene expression, cellular growth, response to DNA damage, of the dek phenotype by the Dsg insertion. The mutation, desig- and nonsense-mediated mRNA decay (9). The protein, called nated dek38-Dsg, is embryonic lethal, has a defective basal endo- Tel2-interacting protein 2 (TTI2), associates with Telomere sperm transfer (BETL) layer, and results in a smaller seed with maintenance 2 (TEL2) and Tel2-interacting protein 1 (TTI1), highly underdeveloped endosperm. The maize dek38 gene en- forming what is called the TTT complex (10). The TTT complex codes a TTI2 (Tel2-interacting protein 2) molecular cochaperone. regulates the steady-state levels of PIKKs, and their assembly In yeast and mammals, TTI2 associates with two other cochaper- into functional complexes in a heat shock protein 90 (HSP90)- ones, TEL2 (Telomere maintenance 2) and TTI1 (Tel2-interacting dependent manner (11). Here, we use the dek38-Dsg mutation to protein 1), to form the triple T complex that regulates DNA dam- report a description of the TTT complex in plants. The mutation age response. Therefore, we cloned the maize Tel2 and Tti1 ho- is lethal and results in an arrested embryo and a highly un- mologs and showed that TEL2 can interact with both TTI1 and derdeveloped endosperm. The mutation also reduces protein TTI2 in yeast two-hybrid assays. The three proteins regulate the levels of two members of maize PIKKs called Target of Rapa- cellular levels of phosphatidylinositol 3-kinase-related kinases mycin (ZmTOR) and Ataxia Telangiectasia Mutated (ZmATM), (PIKKs) and localize to the cytoplasm and the nucleus, consistent suggesting a conserved function throughout eukaryotic species. with known subcellular locations of PIKKs. dek38-Dsg displays re- duced pollen transmission, indicating TTI2’s importance in male Results reproductive cell development. Verification and Genetic Characterization of the dek38-Dsg Mutant. SCIENCES The dek38-Dsg mutant was isolated from a screen of a Dsg in- AGRICULTURAL Ac/Ds | Dsg | Tti2 | TTT complex | PIKKs sertion collection for defective kernel (dek) phenotypes in a predominantly W22 genetic background (Fig. 1). Dsg-adjacent aize has a long history of transposon genetics dating back sequences derived from sequence indexing of this collection Mto the discovery of transposable elements Activator (Ac) and Dissociation (Ds) by Barbara McClintock in the 1940s (1). Significance Transposable elements comprise approximately 85% of the maize genome (2) and are mostly inert because of mutations and Transposable elements (TEs) are important tools to study gene epigenetic silencing by DNA methylation and histone modifica- function in plants, more so than in animal species, because of tions (3, 4). Several of them, however, like the Ac/Ds and Mutator the ease in generating large numbers of germinal transposi- families, can be activated and used for forward or reverse ge- tions. We took advantage of an engineered TE in maize to netics purposes (5–7). The Ac/Ds system is the method of choice broaden this approach. We screened for a defective kernel when performing targeted transposon tagging, but limitations mutant and used the green fluorescent protein encoded by the such as lack of “Ds-launching pads” near the gene of interest, TE to locate the gene linked to the mutation. The property of and identifying germinal excisions and tracking Ds, can be a the TE permitted us to generate revertants and new alleles of challenge. To address these major limitations, a new resource the tagged gene, avoiding complementation assays made has been developed in maize by using an engineered Ds that can cumbersome by difficult transformation protocols in maize. be tracked phenotypically for germinal excision and reinsertion Based on its sequence homology to a cochaperone, the tagged through a change in seed color and green fluorescent protein gene opens a line of research on the role of cochaperones in (GFP) signal (8). The Ds element contains GFP (Dsg) and has seed development. the 5′ and 3′ end sequences that are recognized by Ac for transposition. As such, transpositions from these Dsg launching Author contributions: N.G., H.K.D., and J.M. designed research; N.G. performed research; N.G., Y.L., H.K.D., and J.M. contributed new reagents/analytic tools; N.G., H.K.D., and J.M. pads are also being made and new insertion sites being mapped analyzed data; and N.G., H.K.D., and J.M. wrote the paper. to develop a collection of tagged sites for the maize genetics Reviewers: L.C.H., University of Florida; and C.W., Purdue University. community (www.acdsinsertions.org). The GFP tag makes the Ds The authors declare no conflict of interest. element dominant and, therefore, allows for the phenotypic se- Data deposition: The sequences reported in this paper have been deposited in the Gen- lection of seeds either heterozygous or homozygous for the Bank database (accession nos. KY513721, KY513722, and KY513723). Dsg insertion. 1Present address: Biotechnology Research Institute, Chinese Academy of Agricultural Sci- We have used this system to identify recessive mutations that ences, Beijing 100081, China. prevent normal seed development. Such a screen resulted in the 2To whom correspondence should be addressed. Email: [email protected]. identification of a defective kernel mutant (dek), called dek38- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Dsg. Using the unique Dsg-adjacent sequences, the corresponding 1073/pnas.1703498114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1703498114 PNAS | May 16, 2017 | vol. 114 | no. 20 | 5165–5170 Downloaded by guest on September 24, 2021 Fig. 1. The dek38-Dsg mutant segregating in an ear (A, small kernels) and after shelling (B, Right). When viewed under a blue light (C), segregation between normal non-GFP (+/+), normal GFP (+/−), and dek GFP (−/−) is visible. The change in color of normal kernels not fluorescing GFP is because of exposure to blue light in a dark background. (D, Left) Ear from a dek38 footprint allele with with a frameshift mutation. (D, Right) Ear from the allelism test between the same footprint allele and the dek38-Dsg parent. (E) Same ears as in D, but under blue light. The frameshift allele on left lost Dsg (and fluorescence), but the kernels on the right fluoresce because of the Dsg element in the dek38-Dsg mutant. Also shown are pictures of defective kernels (embryo side up) from the footprint allele (F) and from the allelism test (G). Normal kernels are shown on top for comparison. indicated that the Dsg was inserted in exon 6 of gene model Dsg) and the dek phenotype (0/50 recombinants). Another ad- GRMZM2G048851 in dek38-Dsg, based on mapping sequences vantage is that it is possible to monitor the transmission of the to the maize inbred B73 reference genome assembly version 3 gene (either through male or female) by counting the number of (www.acdsinsertions.org). The Dsg insertion was verified by de- signing PCR primer pairs on the left and right sides of the in- sertion, with one primer designed from the gene model, and the other from the Dsg insertion (Table S1). PCR using these two A GRMZM2G048851 primer pairs gave amplification on the GFP containing Ds ele- Ds ments, but not on non-GFP containing elements. The PCR …CTGGTGGA GFP CTGGTGGA… products were also sequenced and showed that left and right insertion junctions contain an 8-bp direct repeat (Fig. 2), which is D B N D ND WT dek38- characteristic of Ac/Ds transposon insertion sites (12). RT-PCR ZmATM ZmTOR Dsg experiments showed that the Dsg insertion does not affect (approx. 400 kDa) (approx. 250 kDa) Ubiquin Ubiquin transcription of GRMZM2G048851. If translated, the transcript (9 kDa) (9 kDa) is predicted to result in a shorter protein from a premature stop codon, containing amino acid residues from parts of the Dsg se- C N D quence. Protein-folding modeling also predicts a drastic change in GRMZM2048851 27 cycles the 3D conformation of the protein (Fig. 2D). Acn 24 cycles An advantage of having a Dsg-tagged gene is that it is possible to select heterozygotes among the normal seeds in the dek38-Dsg mutant by simple GFP selection. This feature made it easier to Fig. 2. (A) The Dsg is inserted in exon 6 of gene model GRMZM2G048851 conduct a cosegregation analysis between the dek phenotype and (ZmTti2). The 8-bp target site duplication (TSD) sequence is indicated on the left and right sides of the insertion. (B) Western blots comparing ZmATM the Dsg. Selfed progenies from 25 normal GFP seeds segregated and ZmTOR protein levels between normal (N) and dek (D) kernels. (C) Gene for the dek phenotype. Conversely, none of the selfed progenies expression of GRMZM2G048851 between WT and dek using mRNA extracted from 25 normal non-GFP seeds segregated for the dek pheno- from immature seeds.
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