NLRC5 negatively regulates the NF-kappaB and type I interferon signaling pathways.

Stringent control of the NF-kappaB and type I interferon signaling pathways is critical to effective host immune responses, yet the molecular mechanisms that negatively regulate these pathways are poorly understood. Here, we show that NLRC5, a member of the highly conserved NOD-like protein family, can inhibit the IKK complex and RIG-I/MDA5 function. NLRC5 inhibited NF-kappaB-dependent responses by interacting with IKKalpha and IKKbeta and blocking their phosphorylation. It also interacted with RIG-I and MDA5, but not with MAVS, to inhibit RLR-mediated type I interferon responses. Consistent with these observations, NLRC5-specific siRNA knockdown not only enhanced the activation of NF-kappaB and its responsive genes, TNF-alpha and IL-6, but also promoted type I interferon signaling and antiviral immunity. Our findings identify NLRC5 as a negative regulator that blocks two central components of the NF-kappaB and type I interferon signaling pathways and suggest an important role for NLRC5 in homeostatic control of innate immunity.

Molecular and functional analyses of a maize autoactive NB-LRR protein identify precise structural requirements for activity.

Plant disease resistance is often mediated by nucleotide binding-leucine rich repeat (NLR) proteins which remain auto-inhibited until recognition of specific pathogen-derived molecules causes their activation, triggering a rapid, localized cell death called a hypersensitive response (HR). Three domains are recognized in one of the major classes of NLR proteins: a coiled-coil (CC), a nucleotide binding (NB-ARC) and a leucine rich repeat (LRR) domains. The maize NLR gene Rp1-D21 derives from an intergenic recombination event between two NLR genes, Rp1-D and Rp1-dp2 and confers an autoactive HR. We report systematic structural and functional analyses of Rp1 proteins in maize and N. benthamiana to characterize the molecular mechanism of NLR activation/auto-inhibition. We derive a model comprising the following three main features: Rp1 proteins appear to self-associate to become competent for activity. The CC domain is signaling-competent and is sufficient to induce HR. This can be suppressed by the NB-ARC domain through direct interaction. In autoactive proteins, the interaction of the LRR domain with the NB-ARC domain causes de-repression and thus disrupts the inhibition of HR. Further, we identify specific amino acids and combinations thereof that are important for the auto-inhibition/activity of Rp1 proteins. We also provide evidence for the function of MHD2, a previously uncharacterized, though widely conserved NLR motif. This work reports several novel insights into the precise structural requirement for NLR function and informs efforts towards utilizing these proteins for engineering disease resistance.

Correction: Molecular and functional analyses of a maize autoactive NB-LRR protein identify precise structural requirements for activity.

[This corrects the article DOI: 10.1371/journal.ppat.1004674.].

A genome-wide association study of the maize hypersensitive defense response identifies genes that cluster in related pathways.

Much remains unknown of molecular events controlling the plant hypersensitive defense response (HR), a rapid localized cell death that limits pathogen spread and is mediated by resistance (R-) genes. Genetic control of the HR is hard to quantify due to its microscopic and rapid nature. Natural modifiers of the ectopic HR phenotype induced by an aberrant auto-active R-gene (Rp1-D21), were mapped in a population of 3,381 recombinant inbred lines from the maize nested association mapping population. Joint linkage analysis was conducted to identify 32 additive but no epistatic quantitative trait loci (QTL) using a linkage map based on more than 7000 single nucleotide polymorphisms (SNPs). Genome-wide association (GWA) analysis of 26.5 million SNPs was conducted after adjusting for background QTL. GWA identified associated SNPs that colocalized with 44 candidate genes. Thirty-six of these genes colocalized within 23 of the 32 QTL identified by joint linkage analysis. The candidate genes included genes predicted to be in involved programmed cell death, defense response, ubiquitination, redox homeostasis, autophagy, calcium signalling, lignin biosynthesis and cell wall modification. Twelve of the candidate genes showed significant differential expression between isogenic lines differing for the presence of Rp1-D21. Low but significant correlations between HR-related traits and several previously-measured disease resistance traits suggested that the genetic control of these traits was substantially, though not entirely, independent. This study provides the first system-wide analysis of natural variation that modulates the HR response in plants.

Molecular cloning of large alternative transcripts based on comparative phylogenetic analysis and exploration of an EST database.

In animals, a gene may be 50 kb or over and contain multiple alternative transcripts with sequences that are not experimentally validated. Under these special circumstances, PCR-based cloning may become very difficult. Here a simple cloning strategy is described using the mNLRC5 gene as an example. We performed comparative phylogenetic analysis between murine and human NLR protein families to anchor the translation start codon, searched an EST database with the 3' end of the genomic DNA sequence to obtain ESTs from the farthest 3' end of the gene, and isolated the full-length CDS of the mNLRC5 of about 6 kb through conventional RT-PCR and 3' RACE.

WOX4 promotes procambial development.

Plant shoot organs arise from initial cells that are recruited from meristematic tissues. Previous studies have shown that members of the WUSCHEL-related HOMEOBOX (WOX) gene family function to organize various initial cell populations during plant development. The function of the WOX4 gene is previously undescribed in any plant species. Comparative analyses of WOX4 transcription and function are presented in Arabidopsis (Arabidopsis thaliana), a simple-leafed plant with collateral vasculature, and in tomato (Solanum lycopersicum), a dissected-leafed species with bicollateral venation. WOX4 is transcribed in the developing vascular bundles of root and shoot lateral organs in both Arabidopsis and tomato. RNA interference-induced down-regulation of WOX4 in Arabidopsis generated small plants whose vascular bundles accumulated undifferentiated ground tissue and exhibited severe reductions in differentiated xylem and phloem. In situ hybridization analyses of Atwox4-RNA interference plants revealed delayed and reduced expression of both the phloem developmental marker ALTERED PHLOEM1 and HOMEOBOX GENE8, a marker of the vascular procambium. Overexpression of SlWOX4 correlated with overproliferation of xylem and phloem in transgenic tomato seedlings. The cumulative data suggest that the conserved WOX4 function is to promote differentiation and/or maintenance of the vascular procambium, the initial cells of the developing vasculature.

Use of Mutant-Assisted Gene Identification and Characterization (MAGIC) to identify novel genetic loci that modify the maize hypersensitive response.

The partially dominant, autoactive maize disease resistance gene Rp1-D21 causes hypersensitive response (HR) lesions to form spontaneously on leaves and stems in the absence of pathogen recognition. The maize nested association mapping (NAM) population consists of 25 200-line subpopulations each derived from a cross between the maize line B73 and one of 25 diverse inbred lines. By crossing a line carrying the Rp1-D21 gene with lines from three of these subpopulations and assessing the F(1) progeny, we were able to map several novel loci that modify the maize HR, using both single-population quantitative trait locus (QTL) and joint analysis of all three populations. Joint analysis detected QTL in greater number and with greater confidence and precision than did single population analysis. In particular, QTL were detected in bins 1.02, 4.04, 9.03, and 10.03. We have previously termed this technique, in which a mutant phenotype is used as a "reporter" for a trait of interest, Mutant-Assisted Gene Identification and Characterization (MAGIC).

A comprehensive genome variation map of melon identifies multiple domestication events and loci influencing agronomic traits.

Melon is an economically important fruit crop that has been cultivated for thousands of years; however, the genetic basis and history of its domestication still remain largely unknown. Here we report a comprehensive map of the genomic variation in melon derived from the resequencing of 1,175 accessions, which represent the global diversity of the species. Our results suggest that three independent domestication events occurred in melon, two in India and one in Africa. We detected two independent sets of domestication sweeps, resulting in diverse characteristics of the two subspecies melo and agrestis during melon breeding. Genome-wide association studies for 16 agronomic traits identified 208 loci significantly associated with fruit mass, quality and morphological characters. This study sheds light on the domestication history of melon and provides a valuable resource for genomics-assisted breeding of this important crop.

Estimates on compressed neural networks regression.

When the neural element number n of neural networks is larger than the sample size m, the overfitting problem arises since there are more parameters than actual data (more variable than constraints). In order to overcome the overfitting problem, we propose to reduce the number of neural elements by using compressed projection A which does not need to satisfy the condition of Restricted Isometric Property (RIP). By applying probability inequalities and approximation properties of the feedforward neural networks (FNNs), we prove that solving the FNNs regression learning algorithm in the compressed domain instead of the original domain reduces the sample error at the price of an increased (but controlled) approximation error, where the covering number theory is used to estimate the excess error, and an upper bound of the excess error is given.

A connected set of genes associated with programmed cell death implicated in controlling the hypersensitive response in maize.

Rp1-D21 is a maize auto-active resistance gene conferring a spontaneous hypersensitive response (HR) of variable severity depending on genetic background. We report an association mapping strategy based on the Mutant Assisted Gene Identification and Characterization approach to identify naturally occurring allelic variants associated with phenotypic variation in HR. Each member of a collection of 231 diverse inbred lines of maize constituting a high-resolution association mapping panel were crossed to a parental stock heterozygous for Rp1-D21, and the segregating F(1) generation testcrosses were evaluated for phenotypes associated with lesion severity for 2 years at two locations. A genome-wide scan for associations with HR was conducted with 47,445 SNPs using a linear mixed model that controlled for spurious associations due to population structure. Since the ability to identify candidate genes and the resolution of association mapping are highly influenced by linkage disequilibrium (LD), we examined the extent of genome-wide LD. On average, marker pairs separated by >10 kbp had an r(2) value of <0.1. Genomic regions surrounding SNPs significantly associated with HR traits were locally saturated with additional SNP markers to establish local LD structure and precisely identify candidate genes. Six significantly associated SNPs at five loci were detected. At each locus, the associated SNP was located within or immediately adjacent to candidate causative genes predicted to play significant roles in the control of programmed cell death and especially in ubiquitin pathway-related processes.

Restriction site extension PCR: a novel method for high-throughput characterization of tagged DNA fragments and genome walking.

BACKGROUND: Insertion mutant isolation and characterization are extremely valuable for linking genes to physiological function. Once an insertion mutant phenotype is identified, the challenge is to isolate the responsible gene. Multiple strategies have been employed to isolate unknown genomic DNA that flanks mutagenic insertions, however, all these methods suffer from limitations due to inefficient ligation steps, inclusion of restriction sites within the target DNA, and non-specific product generation. These limitations become close to insurmountable when the goal is to identify insertion sites in a high throughput manner. METHODOLOGY/PRINCIPAL FINDINGS: We designed a novel strategy called Restriction Site Extension PCR (RSE-PCR) to efficiently conduct large-scale isolation of unknown genomic DNA fragments linked to DNA insertions. The strategy is a modified adaptor-mediated PCR without ligation. An adapter, with complementarity to the 3' overhang of the endonuclease (KpnI, NsiI, PstI, or SacI) restricted DNA fragments, extends the 3' end of the DNA fragments in the first cycle of the primary RSE-PCR. During subsequent PCR cycles and a second semi-nested PCR (secondary RSE-PCR), touchdown and two-step PCR are combined to increase the amplification specificity of target fragments. The efficiency and specificity was demonstrated in our characterization of 37 tex mutants of Arabidopsis. All the steps of RSE-PCR can be executed in a 96 well PCR plate. Finally, RSE-PCR serves as a successful alternative to Genome Walker as demonstrated by gene isolation from maize, a plant with a more complex genome than Arabidopsis. CONCLUSIONS/SIGNIFICANCE: RSE-PCR has high potential application in identifying tagged (T-DNA or transposon) sequence or walking from known DNA toward unknown regions in large-genome plants, with likely application in other organisms as well.

Analyses of WOX4 transgenics provide further evidence for the evolution of the WOX gene family during the regulation of diverse stem cell functions.

The WOX (WUSCHEL-related homeobox) gene family of Arabidopsis comprises fifteen plant-specific transcriptional factors that play important development roles. Genetic, phylogenetic, and genomic analyses suggest that WOX genes generally act non-autonomously to organize stem-cell and initial-cell populations within plant meristems and organ anlagen. Previous cross-complementation analyses indicate that the functional diversification of distinct WOX paralogs may be explained largely by promoter evolution, although paralog-specific protein::protein interactions are also implicated. A recent report described WOX4 function during development of the procambium, which comprises the meristematic tissues of the plant vasculature. Here we show that WOX4 fails to complement PRS1/WOX3 function, when driven from the PRS1/WOX3 native promoter. These data suggest that WOX4 identifies different DNA targets and/or interacting proteins during development of the vasculature procambium than does PRS1/WOX3 during the specification of lateral organ initial cells. The identification of super-compound leaf phenotypes induced by overexpression of the SlWOX4 ortholog in tomato suggests a functional link between vascular patterning and leaf complexity.

Tissue specificity and evolution of meristematic WOX3 function.

The WUSCHEL-related homeobox (WOX) gene PRESSED FLOWER1 (PRS1) performs a conserved function during lateral organ development in Arabidopsis (Arabidopsis thaliana). Expressed in the periphery of the shoot meristem, PRS1 recruits founder cells that form lateral domains of vegetative and floral organs. Null mutations in PRS1 cause the deletion of lateral stipules from leaves and of lateral sepals and stamens from flowers. Although PRS1 expression is described in the L1 layer, PRS1 recruits founder cells from all meristem layers. The mechanism of non-cell autonomous PRS1 function and the evolution of disparate WOX gene functions are investigated herein. Meristem layer-specific promoters reveal that both L1 and L1-L2 expression of PRS1 fail to fully rescue PRS1 function, and PRS1 protein does not traffic laterally or transversely between shoot meristem layers. PRS1 protein accumulates within all meristematic cell layers (L1-L2-L3) when expressed from the native promoter, presumably due to low-level transcription in the L2 and L3 layers. When driven from the PRS1 promoter, full rescue of vegetative and floral prs1 mutant phenotypes is provided by WUSCHEL1 (WUS1), which is normally expressed in the stem cell organizing center of shoot meristems. The data reveal that WUS1 and PRS1 can engage in equivalent protein-protein interactions and direct transcription of conserved target genes, suggesting that their subfunctionalization has evolved primarily via diverse promoter specificity. Unexpectedly, these results also suggest that meristematic stem cells and lateral organ founder cells are intrinsically similar and formed via equivalent processes such that their ultimate fate is dependent upon stage-specific and domain-specific positional signaling.

The maize duplicate genes narrow sheath1 and narrow sheath2 encode a conserved homeobox gene function in a lateral domain of shoot apical meristems.

The narrow sheath (ns) phenotype of maize is a duplicate factor trait conferred by mutations at the unlinked loci ns1 and ns2. Recessive mutations at each locus together confer the phenotypic deletion of a lateral compartment in maize leaves and leaf homologs. Previous analyses revealed that the mediolateral axis of maize leaves is comprised of at least two distinct compartments, and suggest a model whereby NS function is required to recruit leaf founder cells from a lateral compartment of maize meristems. Genomic clones of two maize homeodomain-encoding genes were isolated by homology to the WUSCHEL-related gene PRESSED FLOWER (PRS). PRS is required for lateral sepal development in Arabidopsis, although no leaf phenotype is reported. Co-segregation of the ns phenotype with multiple mutant alleles of two maize PRS homologs confirms their allelism to ns1 and ns2. Analyses of NS protein accumulation verify that the ns-R mutations are null alleles. ns transcripts are detected in two lateral foci within maize meristems, and in the margins of lateral organ primordia. Whereas ns1 and ns2 transcripts accumulate to equivalent levels in shoot meristems of vegetative seedlings, ns2 transcripts predominate in female inflorescences. Previously undiscovered phenotypes in the pressed flower mutant support a model whereby the morphology of eudicot leaves and monocot grass leaves has evolved via the differential elaboration of upper versus lower leaf zones. A model implicating an evolutionarily conserved NS/PRS function during recruitment of organ founder cells from a lateral domain of plant meristems is discussed.