Matthew J. Moscou

A Simple Cipher Governs DNA Recognition by TAL Effectors

TAL Order Xanthomonas bacteria attack their plant hosts by delivering their own transcription-activator–like (TAL) proteins into the plant cell nucleus and alter the plants gene regulation (see the Perspective by Voytas and Joung). Moscou and Bogdanove (p. 1501, published online 29 October: see the cover) and Boch et al. (p. 1509, published online 29 October) have now discovered how the similar but not identical repeats in the TAL proteins encode the specificity needed for the proteins to find their targets. Each repeat is specific for one DNA base pair, a specificity encoded by hypervariable amino acid positions. Combining several repeats with different amino acids in the hypervariable positions allowed the production of new effectors that targeted new DNA sites. Xanthomonas bacteria use an amino acid–based code to target effector molecules to specific DNA sequences. TAL effectors of plant pathogenic bacteria in the genus Xanthomonas bind host DNA and activate genes that contribute to disease or turn on defense. Target specificity depends on an effector-variable number of typically 34 amino acid repeats, but the mechanism of recognition is not understood. We show that a repeat-variable pair of residues specifies the nucleotides in the target site, one pair to one nucleotide, with no apparent context dependence. Our finding represents a previously unknown mechanism for protein-DNA recognition that explains TAL effector specificity, enables target site prediction, and opens prospects for use of TAL effectors in research and biotechnology.

An Improved Consensus Linkage Map of Barley Based on Flow-Sorted Chromosomes and Single Nucleotide Polymorphism Markers

Recent advances in high‐throughput genotyping have made it easier to combine information from different mapping populations into consensus genetic maps, which provide increased marker density and genome coverage compared to individual maps. Previously, a single nucleotide polymorphism (SNP)‐based genotyping platform was developed and used to genotype 373 individuals in four barley (Hordeum vulgare L.) mapping populations. This led to a 2943 SNP consensus genetic map with 975 unique positions. In this work, we add data from six additional populations and more individuals from one of the original populations to develop an improved consensus map from 1133 individuals. A stringent and systematic analysis of each of the 10 populations was performed to achieve uniformity. This involved reexamination of the four populations included in the previous map. As a consequence, we present a robust consensus genetic map that contains 2994 SNP loci mapped to 1163 unique positions. The map spans 1137.3 cM with an average density of one marker bin per 0.99 cM. A novel application of the genotyping platform for gene detection allowed the assignment of 2930 genes to flow‐sorted chromosomes or arms, confirmed the position of 2545 SNP‐mapped loci, added chromosome or arm allocations to an additional 370 SNP loci, and delineated pericentromeric regions for chromosomes 2H to 7H. Marker order has been improved and map resolution has been increased by almost 20%. These increased precision outcomes enable more optimized SNP selection for marker‐assisted breeding and support association genetic analysis and map‐based cloning. It will also improve the anchoring of DNA sequence scaffolds and the barley physical map to the genetic map.

Engineering Plant Disease Resistance Based on TAL Effectors

Transcription activator-like (TAL) effectors are encoded by plant-pathogenic bacteria and induce expression of plant host genes. TAL effectors bind DNA on the basis of a unique code that specifies binding of amino acid residues in repeat units to particular DNA bases in a one-to-one correspondence. This code can be used to predict binding sites of natural TAL effectors and to design novel synthetic DNA-binding domains for targeted genome manipulation. Natural mechanisms of resistance in plants against TAL effector-containing pathogens have given insights into new strategies for disease control.

Strategies for transferring resistance into wheat: from wide crosses to GM cassettes

The domestication of wheat in the Fertile Crescent 10,000 years ago led to a genetic bottleneck. Modern agriculture has further narrowed the genetic base by introducing extreme levels of uniformity on a vast spatial and temporal scale. This reduction in genetic complexity renders the crop vulnerable to new and emerging pests and pathogens. The wild relatives of wheat represent an important source of genetic variation for disease resistance. For nearly a century farmers, breeders, and cytogeneticists have sought to access this variation for crop improvement. Several barriers restricting interspecies hybridization and introgression have been overcome, providing the opportunity to tap an extensive reservoir of genetic diversity. Resistance has been introgressed into wheat from at least 52 species from 13 genera, demonstrating the remarkable plasticity of the wheat genome and the importance of such natural variation in wheat breeding. Two main problems hinder the effective deployment of introgressed resistance genes for crop improvement: (1) the simultaneous introduction of genetically linked deleterious traits and (2) the rapid breakdown of resistance when deployed individually. In this review, we discuss how recent advances in molecular genomics are providing new opportunities to overcome these problems.

Quantitative and qualitative stem rust resistance factors in barley are associated with transcriptional suppression of defense regulons

Stem rust (Puccinia graminis f. sp. tritici; Pgt) is a devastating fungal disease of wheat and barley. Pgt race TTKSK (isolate Ug99) is a serious threat to these Triticeae grain crops because resistance is rare. In barley, the complex Rpg-TTKSK locus on chromosome 5H is presently the only known source of qualitative resistance to this aggressive Pgt race. Segregation for resistance observed on seedlings of the Q21861 × SM89010 (QSM) doubled-haploid (DH) population was found to be predominantly qualitative, with little of the remaining variance explained by loci other than Rpg-TTKSK. In contrast, analysis of adult QSM DH plants infected by field inoculum of Pgt race TTKSK in Njoro, Kenya, revealed several additional quantitative trait loci that contribute to resistance. To molecularly characterize these loci, Barley1 GeneChips were used to measure the expression of 22,792 genes in the QSM population after inoculation with Pgt race TTKSK or mock-inoculation. Comparison of expression Quantitative Trait Loci (eQTL) between treatments revealed an inoculation-dependent expression polymorphism implicating Actin depolymerizing factor3 (within the Rpg-TTKSK locus) as a candidate susceptibility gene. In parallel, we identified a chromosome 2H trans-eQTL hotspot that co-segregates with an enhancer of Rpg-TTKSK-mediated, adult plant resistance discovered through the Njoro field trials. Our genome-wide eQTL studies demonstrate that transcript accumulation of 25% of barley genes is altered following challenge by Pgt race TTKSK, but that few of these genes are regulated by the qualitative Rpg-TTKSK on chromosome 5H. It is instead the chromosome 2H trans-eQTL hotspot that orchestrates the largest inoculation-specific responses, where enhanced resistance is associated with transcriptional suppression of hundreds of genes scattered throughout the genome. Hence, the present study associates the early suppression of genes expressed in this host–pathogen interaction with enhancement of R-gene mediated resistance.

Nonhost resistance to rust pathogens – a continuation of continua

The rust fungi (order: Pucciniales) are a group of widely distributed fungal plant pathogens, which can infect representatives of all vascular plant groups. Rust diseases significantly impact several crop species and considerable research focuses on understanding the basis of host specificity and nonhost resistance. Like many pathogens, rust fungi vary considerably in the number of hosts they can infect, such as wheat leaf rust (Puccinia triticina), which can only infect species in the genera Triticum and Aegilops, whereas Asian soybean rust (Phakopsora pachyrhizi) is known to infect over 95 species from over 42 genera. A greater understanding of the genetic basis determining host range has the potential to identify sources of durable resistance for agronomically important crops. Delimiting the boundary between host and nonhost has been complicated by the quantitative nature of phenotypes in the transition between these two states. Plant–pathogen interactions in this intermediate state are characterized either by (1) the majority of accessions of a species being resistant to the rust or (2) the rust only being able to partially complete key components of its life cycle. This leads to a continuum of disease phenotypes in the interaction with different plant species, observed as a range from compatibility (host) to complete immunity within a species (nonhost). In this review we will highlight how the quantitative nature of disease resistance in these intermediate interactions is caused by a continuum of defense barriers, which a pathogen needs to overcome for successfully establishing itself in the host. To illustrate continua as this underlying principle, we will discuss the advances that have been made in studying nonhost resistance towards rust pathogens, particularly cereal rust pathogens.

Infection of Brachypodium distachyon with Selected Grass Rust Pathogens

The model temperate grass Brachypodium distachyon is considered a nonhost for wheat rust diseases caused by Puccinia graminis f. sp. tritici, P. triticina, and P. striiformis. Up to 140 Brachypodium accessions were infected with these three rust species, in addition to P. graminis ff. spp. avena and phalaridis. Related B. distachyon lines showed similar cytological nonhost resistance (NHR) phenotypes, and an inverse relationship between P. graminis f. sp. tritici and P. striiformis growth was observed in many lines, with accessions that allowed the most growth of P. graminis f. sp. tritici showing the least P. striiformis development and vice versa. Callose deposition patterns during infection by all three rust species showed similarity to the wheat basal defense response while cell death that resulted in autofluorescence did not appear to be a major component of the defense response. Infection of B. distachyon with P. graminis f. sp. avena and P. graminis f. sp. phalaridis produced much greater colonization, indicating that P. graminis rusts with Poeae hosts show greater ability to infect B. distachyon than those with Triticeae hosts. P. striiformis infection of progeny from two B. distachyon families demonstrated that these NHR phenotypes are highly heritable and appear to be under relatively simple genetic control, making this species a powerful tool for elucidating the molecular basis of NHR to cereal rust pathogens.

Sequencing of 15 622 gene-bearing BACs clarifies the gene-dense regions of the barley genome

Summary Barley (Hordeum vulgare L.) possesses a large and highly repetitive genome of 5.1 Gb that has hindered the development of a complete sequence. In 2012, the International Barley Sequencing Consortium released a resource integrating whole‐genome shotgun sequences with a physical and genetic framework. However, because only 6278 bacterial artificial chromosome (BACs) in the physical map were sequenced, fine structure was limited. To gain access to the gene‐containing portion of the barley genome at high resolution, we identified and sequenced 15 622 BACs representing the minimal tiling path of 72 052 physical‐mapped gene‐bearing BACs. This generated ~1.7 Gb of genomic sequence containing an estimated 2/3 of all Morex barley genes. Exploration of these sequenced BACs revealed that although distal ends of chromosomes contain most of the gene‐enriched BACs and are characterized by high recombination rates, there are also gene‐dense regions with suppressed recombination. We made use of published map‐anchored sequence data from Aegilops tauschii to develop a synteny viewer between barley and the ancestor of the wheat D‐genome. Except for some notable inversions, there is a high level of collinearity between the two species. The software HarvEST:Barley provides facile access to BAC sequences and their annotations, along with the barley–Ae. tauschii synteny viewer. These BAC sequences constitute a resource to improve the efficiency of marker development, map‐based cloning, and comparative genomics in barley and related crops. Additional knowledge about regions of the barley genome that are gene‐dense but low recombination is particularly relevant.

Quantitative and temporal definition of the Mla transcriptional regulon during barley-powdery mildew interactions.

Barley Mildew resistance locus a (Mla) is a major determinant of immunity to the powdery mildew pathogen, Blumeria graminis f. sp. hordei. Alleles of Mla encode cytoplasmic- and membrane-localized coiled-coil, nucleotide binding site, leucine-rich repeat proteins that mediate resistance when complementary avirulence effectors (AVR(a)) are present in the pathogen. Presence of an appropriate AVR(a) protein triggers nuclear relocalization of MLA, in which MLA binds repressing host transcription factors. Timecourse expression profiles of plants harboring Mla1, Mla6, and Mla12 wild-type alleles versus paired loss-of-function mutants were compared to discover conserved transcriptional targets of MLA and downstream signaling cascades. Pathogen-dependent gene expression was equivalent or stronger in susceptible plants at 20 h after inoculation (HAI) and was attenuated at later timepoints, whereas resistant plants exhibited a time-dependent strengthening of the transcriptional response, increasing in both fold change and the number of genes differentially expressed. Deregulation at 20 HAI implicated 16 HAI as a crucial point in determining the future trajectory of this interaction and was interrogated by quantitative analysis. In total, 28 potential transcriptional targets of the MLA regulon were identified. These candidate targets possess a diverse set of predicted functions, suggesting that multiple pathways are required to mediate the hypersensitive reaction.

Quantitative Genetic Dissection of Shoot Architecture Traits in Maize: Towards a Functional Genomics Approach

Quantitative trait loci (QTL) affecting the total number of leaves made before flowering and the number of leaves below the uppermost ear (NLBE) were mapped and characterized using the intermated B73 × Mo17 recombinant inbred lines (IBMRILs) of maize (Zea mays L.). B73 and Mo17 typically make 20 and 17 leaves, 14 and 11 of which are below the ear. Total number of leaves and the number of leaves below the uppermost ear are ∼80% heritable in the IBMRILs, which show strongly transgressive phenotypic ranges of 15 to 24 and 10 to 18 leaves for these traits. B73 alleles at loci in chromosome bins 1.06, 3.06, 4.08, 8.04, 8.05, 9.07, and 10.04 increase leaf numbers, with all but the 3.06 QTL affecting both of these highly correlated traits (r = 0.86, p < 0.0001). Conservative QTL confidence intervals were computed and projected onto the draft maize genome sequence, revealing very narrow localizations (∼1 Mb) for four of the seven loci. More than 40% of the heritable variation for both traits is explained by an additive model, squarely accounting for the dramatic parental differences, but leaving the basis of the strong transgression unexplained. In addition, error rate control and confidence interval methods tailored for composite interval mapping are introduced, and their potential for improving QTL reporting is discussed.