Evidence for a natural allelic series at the Maize domestication Locus teosinte branched1

Despite numerous quantitative trait loci and association mapping studies, our understanding of the extent to which natural allelic series contribute to the variation for complex traits is limited. In this study, we investigate the occurrence of a natural allelic series for complex traits at the teosinte branched1 (tb1) gene in natural populations of teosinte (Zea mays ssp. parviglumis, Z. mays ssp. mexicana, and Z. diploperennis). Previously, tb1 was shown to confer large effects on both plant architecture and ear morphology between domesticated maize and teosinte; however, the effect of tb1 on trait variation in natural populations of teosinte has not been investigated. We compare the effects of nine teosinte alleles of tb1 that were introgressed into an isogenic maize inbred background. Our results provide evidence for a natural allelic series at tb1 for several complex morphological traits. The teosinte introgressions separate into three distinct phenotypic classes, which correspond to the taxonomic origin of the alleles. The effects of the three allelic classes also correspond to known morphological differences between the teosinte taxa. Our results suggest that tb1 contributed to the morphological diversification of teosinte taxa as well as to the domestication of maize.     

Novel Interallelic Complementation at the his1 Locus of Yeast

In yeast, 17 histidine-requiring mutants derived from and interallelically complementary to his1-7 were analyzed. The genetic basis of the complementation response was elucidated by mitotic and meiotic gene conversion. Each allele probably carries an unaltered 7-site mutation and a unique second-site alteration. The second-site alterations appear to be clustered within the proximal and distal segments of the his1 structural gene. Models of intraalelic complementation are reviewed in light of the unique complementational response between a single-site mutant and a double mutant including the identical altered base sequence.     

Maize Unstable factor for orange1 Is Required for Maintaining Silencing Associated with Paramutation at the pericarp color1 and booster1 Loci

To understand the molecular mechanisms underlying paramutation, we examined the role of Unstable factor for orange1 (Ufo1) in maintaining paramutation at the maize pericarp color1 (p1) and booster1 (b1) loci. Genetic tests revealed that the Ufo1-1 mutation disrupted silencing associated with paramutation at both p1 and b1. The level of up regulation achieved at b1 was lower than that at p1, suggesting differences in the role Ufo1-1 plays at these loci. We characterized the interaction of Ufo1-1 with two silenced p1 epialleles, P1-rr′ and P1-pr^TP, that were derived from a common P1-rr ancestor. Both alleles are phenotypically indistinguishable, but differ in their paramutagenic activity; P1-rr′ is paramutagenic to P1-rr, while P1-pr^TP is non-paramutagenic. Analysis of cytosine methylation revealed striking differences within an enhancer fragment that is required for paramutation; P1-rr′ exhibited increased methylation at symmetric (CG and CHG) and asymmetric (CHH) sites, while P1-pr^TP was methylated only at symmetric sites. Both silenced alleles had higher levels of dimethylation of lysine 9 on histone 3 (H3K9me2), an epigenetic mark of silent chromatin, in the enhancer region. Both epialleles were reactivated in the Ufo1-1 background; however, reactivation of P1-rr′ was associated with dramatic loss of symmetric and asymmetric cytosine methylation in the enhancer, while methylation of up-regulated P1-pr^TP was not affected. Interestingly, Ufo1-1–mediated reactivation of both alleles was accompanied with loss of H3K9me2 mark from the enhancer region. Therefore, while earlier studies have shown correlation between H3K9me2 and DNA methylation, our study shows that these two epigenetic marks are uncoupled in the Ufo1-1–reactivated p1 alleles. Furthermore, while CHH methylation at the enhancer region appears to be the major distinguishing mark between paramutagenic and non-paramutagenic p1 alleles, H3K9me2 mark appears to be important for maintaining epigenetic silencing.     

Insertions of a Novel Class of Transposable Elements with a Strong Target Site Preference at the R Locus of Maize

The r locus of maize regulates anthocyanin synthesis in various tissues of maize through the production of helix-loop-helix DNA binding proteins capable of inducing expression of structural genes in the anthocyanin biosynthetic pathway. The complex r variant, R-r:standard (R-r), undergoes frequent mutation through a variety of mechanisms including displaced synapsis and crossing over, and intrachromosomal recombination. Here we report a new mechanism for mutation at the R-r complex: insertion of a novel family of transposable elements. Because the elements were first identified in the R-p gene of the R-r complex, they have been named P Instability Factor (PIF). Two different PIF elements were cloned and found to have identical sequences at their termini but divergent internal sequences. In addition, the PIF elements showed a marked specificity of insertion sites. Six out of seven PIF-containing derivatives examined had an element inserted at an identical location. Two different members of the PIF element family were identified at this position. The seventh PIF-containing derivative examined had the element inserted at a distinct position within r. Even at this location, however, the element inserted into a conserved target sequence. The timing of PIF excision is unusual. Germinal excision rates can range up to several percent of progeny. Yet somatic sectors are rare, even in lines exhibiting high germinal reversion rates.     

Ds-Induced Alleles at the OPAQUE-2 Locus of Maize

Transposon mutagenesis has been used to isolate mutable alleles at the Opaque-2 (O2) locus of maize. Plants with the Activator-Dissociation (Ac-Ds) system of transposable elements and O2 were crossed as males to a stable o2 tester line. Among a population of 200,000 kernels, 198 exceptional kernels with somatic instability were recovered. In four cases, designated O2-m1, o2-m2, O2-m3 and O2-m4, variegated phenotypes appeared in F₂ and subsequent generations. Genetic analyses indicated that the presence of Ds near or within the O2 gene was responsible for the observed somatic instability at the O2 locus. The phenotypes of the newly induced alleles were of two types. Alleles O2-m1, O2-m3 and O2-m4, in the absence of Ac, were characterized by kernel phenotypes indistinguishable from the wild type; in the presence of Ac they generated kernels with opaque sectors interspersed within a vitreous background. In contrast, the mutable allele o2-m2, in the absence of Ac, was characterized by kernels with a recessive phenotype similar to o2 recessive mutants. In the presence of Ac, it reverted somatically to wild-type-producing kernels with vitreous spots in an o2 background. The association of the Ds element with the O2 locus may prove a valuable tool directed to the isolation of DNA fragments bearing the O2 gene.     

Ac Induces Homologous Recombination at the Maize P Locus

The maize P gene conditions red phlobaphene pigmentation to the pericarp and cob. Starting from two unstable P alleles which carry insertions of the transposable element Ac, we have derived 51 P null alleles; 47 of the 51 null alleles have a 17-kb deletion which removes the 4.5-kb Ac element and 12.5 kb of P sequences flanking both sides of Ac. The deletion endpoints lie within two 5.2-kb homologous direct repeats which flank the P gene. A P allele which contains the direct repeats, but does not have an Ac insertion between the direct repeats, shows very little sporophytic or gametophytic instability. The apparent frequency of sporophytic mutations was not increased when Ac was introduced in trans. Southern analysis of DNA prepared from the pericarp tissue demonstrates that the deletions can occur premeiotically, in the somatic cells during development of the pericarp. Evidence is presented that the deletions occurred by homologous recombination between the two direct repeats, and that the presence of an Ac element at the P locus is associated with the recombination/deletion. These results add another aspect to the spectrum of activities of Ac: the destabilization of flanking direct repeat sequences.     

Unraveling Genomic Complexity at a Quantitative Disease Resistance Locus in Maize

Multiple disease resistance has important implications for plant fitness, given the selection pressure that many pathogens exert directly on natural plant populations and indirectly via crop improvement programs. Evidence of a locus conditioning resistance to multiple pathogens was found in bin 1.06 of the maize genome with the allele from inbred line “Tx303” conditioning quantitative resistance to northern leaf blight (NLB) and qualitative resistance to Stewart’s wilt. To dissect the genetic basis of resistance in this region and to refine candidate gene hypotheses, we mapped resistance to the two diseases. Both resistance phenotypes were localized to overlapping regions, with the Stewart’s wilt interval refined to a 95.9-kb segment containing three genes and the NLB interval to a 3.60-Mb segment containing 117 genes. Regions of the introgression showed little to no recombination, suggesting structural differences between the inbred lines Tx303 and “B73,” the parents of the fine-mapping population. We examined copy number variation across the region using next-generation sequencing data, and found large variation in read depth in Tx303 across the region relative to the reference genome of B73. In the fine-mapping region, association mapping for NLB implicated candidate genes, including a putative zinc finger and pan1. We tested mutant alleles and found that pan1 is a susceptibility gene for NLB and Stewart’s wilt. Our data strongly suggest that structural variation plays an important role in resistance conditioned by this region, and pan1, a gene conditioning susceptibility for NLB, may underlie the QTL.     

Interallelic Complementation at the sh Locus in Maize at the Enzyme Level

EMS-induced sh mutants and their heterozygotes were examined for the enzyme, sucrose synthetase, which has previously been shown to be coded by the Sh locus. Complementing heterozygotes have a wild-type phenotype, but show no hybrid protein band after starch gel electrophoresis. The existence of a heteromeric complex, however, is inferred from the two-fold elevation in sucrose cleavage activity in the complementing heterozygotes as compared to the mutant homozygotes. The observations on complementation described here are unique, as the elevation in the activity of this reversible enzyme is noticed only in one direction (viz, sucrose cleavage) of the reaction and not the other (sucrose synthesis).     

Identification of the Maize Gravitropism Gene lazy plant1 by a Transposon-Tagging Genome Resequencing Strategy

Since their initial discovery, transposons have been widely used as mutagens for forward and reverse genetic screens in a range of organisms. The problems of high copy number and sequence divergence among related transposons have often limited the efficiency at which tagged genes can be identified. A method was developed to identity the locations of Mutator (Mu) transposons in the Zea mays genome using a simple enrichment method combined with genome resequencing to identify transposon junction fragments. The sequencing library was prepared from genomic DNA by digesting with a restriction enzyme that cuts within a perfectly conserved motif of the Mu terminal inverted repeats (TIR). Paired-end reads containing Mu TIR sequences were computationally identified and chromosomal sequences flanking the transposon were mapped to the maize reference genome. This method has been used to identify Mu insertions in a number of alleles and to isolate the previously unidentified lazy plant1 (la1) gene. The la1 gene is required for the negatively gravitropic response of shoots and mutant plants lack the ability to sense gravity. Using bioinformatic and fluorescence microscopy approaches, we show that the la1 gene encodes a cell membrane and nuclear localized protein. Our Mu-Taq method is readily adaptable to identify the genomic locations of any insertion of a known sequence in any organism using any sequencing platform.     

Identification and Fine Mapping of rhm1 Locus for Resistance to Southern Corn Leaf Blight in Maize

rhm1 is a major recessive disease resistance locus for Southern corn leaf blight (SCLB).To further narrow down its genetic position,F 2 population and BC 1 F 1 population derived from the cross between resistant (H95 rhm) and susceptible parents (H95) of maize (Zea mays) were constructed.Using newly developed markers,rhm1 was initially delimited within an interval of 2.5 Mb,and then finally mapped to a 8.56 kb interval between InDel marker IDP961-503 and simple sequence repeat (SSR) marker A194149-1.Three polymorphic markers IDP961-504,IDP B2-3 and A194149-2 were shown to be co-segregated with the rhm1 locus.Sequence analysis of the 8.56 kb DNA fragment revealed that it contained only one putative gene with a predicted amino acid sequence identical to lysine histidine transporter 1 (LHT1).Comparative sequence analysis indicated that the LHT1 in H95 rhm harbors a 354 bp insertion in its third exon as compared with that of susceptible alleles in B73,H95 and Mo17.The 354 bp insertion resulted in a truncation of the predicted protein of candidate resistance allele (LHT1-H95 rhm).Our results strongly suggest LHT1 as the candidate gene for rhm1 against SCLB.The tightly linked molecular markers developed in this study can be directly used for molecular breeding of resistance to Southern corn leaf blight in maize.     

Identification of a Maize Locus That Modulates the Hypersensitive Defense Response,Using Mutant-Assisted Gene Identification and Characterization

Potentially useful naturally occurring genetic variation is often difficult to identify as the effects of individual genes are subtle and difficult to observe. In this study, a novel genetic technique called Mutant-Assisted Gene Identification and Characterization is used to identify naturally occurring loci modulating the hypersensitive defense response (HR) in maize. Mutant-Assisted Gene Identification and Characterization facilitates the identification of naturally occurring alleles underlying phenotypic variation from diverse germplasm, using a mutant phenotype as a “reporter.” In this study the reporter phenotype was caused by a partially dominant autoactive disease resistance gene, Rp1-D21, which caused HR lesions to form spontaneously all over the plant. Here it is demonstrated that the Rp1-D21 phenotype is profoundly affected by genetic background. By crossing the Rp1-D21 gene into the IBM mapping population, it was possible to map and identify Hrml1 on chromosome 10, a locus responsible for modulating the HR phenotype conferred by Rp1-D21. Other loci with smaller effects were identified on chromosomes 1 and 9. These results demonstrate that Mutant-Assisted Gene Identification and Characterization is a viable approach for identifying naturally occurring useful genetic variation.POTENTIALLY useful naturally occurring genetic variation is often difficult to identify as the effects of individual genes are subtle and difficult to observe. Furthermore, so many different alleles are available that it is a major challenge just to sift through the enormous diversity available. To this end, we recently conceptualized a simple yet effective method to discover and characterize variation present naturally in plant germplasm (Johal et al. 2008). This method, Mutant-Assisted Gene Identification and Characterization, makes use of a mutant phenotype for a gene affecting the trait of interest as a reporter to discover and analyze relevant, interacting genes present naturally in diverse germplasm. Mutant-Assisted Gene Identification and Characterization involves crossing a mutant to diverse germplasm and then evaluating the mutant progeny for transgressive changes (both suppressed and severe) in the mutant phenotype(s). If the mutation is recessive, the population needs to be advanced to the F₂ generation to be able to detect and analyze such variation. However, for a dominant or partially dominant mutant, evaluations can be made immediately in the F₁ to discover lines that contain suppressors or enhancers of the trait (mutation) under study. Mutant F₁ progenies from such crosses can then be propagated further to identify, map, and clone genes/QTL that affect the trait positively or negatively. In the case of maize and other species for which genetically characterized mapping populations are available, modifying loci can be rapidly mapped by crossing a mutant line to each member of a mapping population and evaluating the resulting F₁ families. In this study we provide a proof-of-concept for the Mutant-Assisted Gene Identification and Characterization technique, using it to identify loci involved in the defense response of maize.Plants are constantly exposed to numerous potential pathogens with diverse modes of attack. Nevertheless, it is rather rare to see plants succumbing to disease. One key reason for this is the presence of a highly effective and inducible defense system, a major component of which is the hypersensitive response (HR). HR is usually associated with a specific recognition event and is activated after other nonspecific resistance mechanisms have been overcome or evaded (see Bent and Mackey 2007). Although it was initially coined to refer to the rapid collapse of cells at the site of infection, over the years the term HR has been used to refer to both cell death and the associated induction of a number of other defense responses, including the accumulation of phytoalexins and pathogenesis-related (PR) proteins at the site of infection, to name a few (Mur et al. 2007). Reactive oxygen species such as superoxide and H₂O₂ appear to be causally involved in cell death underlying the HR response (Jones and Dangl 2006).HR is under the control of a subset of disease-resistance genes, commonly referred to as R genes. These R genes specifically recognize matching avirulence (Avr) effectors from the pathogen. Many R genes encode products containing a nucleotide-binding site (NBS) domain in the middle of the protein and a leucine-rich repeat (LRR) domain at the C-terminal end (Bent and Mackey 2007). R proteins are involved both in the recognition of the pathogen and the subsequent induction of the HR response. How R proteins remain in a quiescent but “vigilant” state remains to be established. Certain mutations in R genes have been found that abolish their dependence on AVR proteins for activation. Such aberrant R genes mostly behave as dominant or partially dominant alleles and trigger the HR constitutively in the absence of the pathogen (Hu et al. 1996; Zhang et al. 2003; Dodds et al. 2006). Two consequences of such “autoactive” or “ectopically active” R genes are a massive induction of cell death and the consequential stunting of the organism (Dodds et al. 2006). Although autoactive R genes have been found to exist in many plant species, the first few examples came from the maize Rp1 locus, which confers race-specific resistance to common rust, caused by Puccinia sorghi (Hu et al. 1996). Such autoactive R genes can be used to investigate HR genetics and etiology in the absence of confounding effects from the pathogen and constitute an excellent candidate for analysis using Mutant-Assisted Gene Identification and Characterization.The details of the HR cell death reaction as well as the pathway(s) that link R gene activation with the HR remain unclear (Mur et al. 2007). Despite considerable research over the past decade, only a few components have been found thus far. Some of these, Ndr1, Eds1, Pad4, Rar1, and Sgt1, were identified in mutagenesis screens conducted to identify mutants that failed to undergo an HR reaction in response to infection by an avirulent pathogen (reviewed in Bent and Mackey 2007). A few others, RIN4, for example, were identified in yeast two-hybrid assays using an NBS–LRR protein as bait (Mackey et al. 2003). Recently, an Arabidopsis gain-of-function mutant that carries a point mutation in an R gene analog (a gene with the structure of an R gene but not known to be involved in resistance to any pathogen) was used to isolate a few more potential genes in the HR pathway in a second site suppressor approach following mutagenesis with ethane methyl sulfonate (EMS) (Palma et al. 2005; Zhang and Li 2005; Goritschnig et al. 2007). A problem with approaches based on intentional mutagenesis is that they fail to uncover genes that have either redundant or essential functions. One way to avoid this problem would be to seek naturally occurring allelic variants affecting HR. Such natural variation is pervasive in all species, being generated and selected for over millions of years of evolution.Although natural variation has served as a constant provider of the R genes in all plant species, natural variability has not been tapped as a tool for understanding other aspects of the disease-resistance response (Holub 2007). The Rp1-D21 gene is an autoactive allele from the maize Rp1 disease-resistance locus that initiates HR randomly all over the plant (Pryor 1993; Collins et al. 1999; Sun et al. 2001). Our objective for this study was to use the Rp1-D21 gene phenotype as a test case for the Mutant-Assisted Gene Identification and Characterization approach. We show here that enormous variation exists in the maize germplasm that is capable of affecting the HR response positively or negatively and we identify loci that modulate expression of the HR phenotype segregating in the well-known Intermated B73 × Mo17 (IBM) advanced intercross line (AIL) population (Coe et al. 2002; Lee et al. 2002). This constitutes the first demonstration of the utility of the Mutant-Assisted Gene Identification and Characterization approach—an approach that is likely to prove widely applicable.     

Identification of a Novel Structural Protein of Arteriviruses

Arteriviruses are positive-stranded RNA viruses with an efficiently organized, polycistronic genome. A short region between the replicase gene and open reading frame (ORF) 2 of the equine arteritis virus (EAV) genome was previously assumed to be untranslated. However, here we report that this segment of the EAV genome contains the 5' part of a novel gene (ORF 2a) which is conserved in all arteriviruses. The 3' part of EAV ORF 2a overlaps with the 5' part of the former ORF 2 (now renamed ORF 2b), which encodes the GS glycoprotein. Both ORF 2a and ORF 2b appear to be expressed from mRNA 2, which thereby constitutes the first proven example of a bicistronic mRNA in arteriviruses. The 67-amino-acid protein encoded by EAV ORF 2a, which we have provisionally named the envelope (E) protein, is very hydrophobic and has a basic C terminus. An E protein-specific antiserum was raised and used to demonstrate the expression of the novel gene in EAV-infected cells. The EAV E protein proved to be very stable, did not form disulfide-linked oligomers, and was not N-glycosylated. Immunofluorescence and immunoelectron microscopy studies showed that the E protein associates with intracellular membranes both in EAV-infected cells and upon independent expression. An analysis of purified EAV particles revealed that the E protein is a structural protein. By using reverse genetics, we demonstrated that both the EAV E and GS proteins are essential for the production of infectious progeny virus.     

Identification of the Kelch Family Protein Nd1-L as a Novel Molecular Interactor of KRIT1

Loss-of-function mutations of the KRIT1 gene (CCM1) have been associated with the Cerebral Cavernous Malformation (CCM) disease, which is characterized by serious alterations of brain capillary architecture. The KRIT1 protein contains multiple interaction domains and motifs, suggesting that it might act as a scaffold for the assembly of functional protein complexes involved in signaling networks. In previous work, we defined structure-function relationships underlying KRIT1 intramolecular and intermolecular interactions and nucleocytoplasmic shuttling, and found that KRIT1 plays an important role in molecular mechanisms involved in the maintenance of the intracellular Reactive Oxygen Species (ROS) homeostasis to prevent oxidative cellular damage. Here we report the identification of the Kelch family protein Nd1-L as a novel molecular interactor of KRIT1. This interaction was discovered through yeast two-hybrid screening of a mouse embryo cDNA library, and confirmed by pull-down and co-immunoprecipitation assays of recombinant proteins, as well as by co-immunoprecipitation of endogenous proteins in human endothelial cells. Furthermore, using distinct KRIT1 isoforms and mutants, we defined the role of KRIT1 domains in the Nd1-L/KRIT1 interaction. Finally, functional assays showed that Nd1-L may contribute to the regulation of KRIT1 nucleocytoplasmic shuttling and cooperate with KRIT1 in modulating the expression levels of the antioxidant protein SOD2, opening a novel avenue for future mechanistic studies. The identification of Nd1-L as a novel KRIT1 interacting protein provides a novel piece of the molecular puzzle involving KRIT1 and suggests a potential functional cooperation in cellular responses to oxidative stress, thus expanding the framework of molecular complexes and mechanisms that may underlie the pathogenesis of CCM disease.