Network Walking charts transcriptional dynamics of nitrogen signaling by integrating validated and predicted genome-wide interactions.

Charting a temporal path in gene networks requires linking early transcription factor (TF)-triggered events to downstream effects. We scale-up a cell-based TF-perturbation assay to identify direct regulated targets of 33 nitrogen (N)-early response TFs encompassing 88% of N-responsive Arabidopsis genes. We uncover a duality where each TF is an inducer and repressor, and in vitro cis-motifs are typically specific to regulation directionality. Validated TF-targets (71,836) are used to refine precision of a time-inferred root network, connecting 145 N-responsive TFs and 311 targets. These data are used to chart network paths from direct TF1-regulated targets identified in cells to indirect targets responding only in planta via Network Walking. We uncover network paths from TGA1 and CRF4 to direct TF2 targets, which in turn regulate 76% and 87% of TF1 indirect targets in planta, respectively. These results have implications for N-use and the approach can reveal temporal networks for any biological system.

Temporal transcriptional logic of dynamic regulatory networks underlying nitrogen signaling and use in plants.

This study exploits time, the relatively unexplored fourth dimension of gene regulatory networks (GRNs), to learn the temporal transcriptional logic underlying dynamic nitrogen (N) signaling in plants. Our "just-in-time" analysis of time-series transcriptome data uncovered a temporal cascade of cis elements underlying dynamic N signaling. To infer transcription factor (TF)-target edges in a GRN, we applied a time-based machine learning method to 2,174 dynamic N-responsive genes. We experimentally determined a network precision cutoff, using TF-regulated genome-wide targets of three TF hubs (CRF4, SNZ, and CDF1), used to "prune" the network to 155 TFs and 608 targets. This network precision was reconfirmed using genome-wide TF-target regulation data for four additional TFs (TGA1, HHO5/6, and PHL1) not used in network pruning. These higher-confidence edges in the GRN were further filtered by independent TF-target binding data, used to calculate a TF "N-specificity" index. This refined GRN identifies the temporal relationship of known/validated regulators of N signaling (NLP7/8, TGA1/4, NAC4, HRS1, and LBD37/38/39) and 146 additional regulators. Six TFs-CRF4, SNZ, CDF1, HHO5/6, and PHL1-validated herein regulate a significant number of genes in the dynamic N response, targeting 54% of N-uptake/assimilation pathway genes. Phenotypically, inducible overexpression of CRF4 in planta regulates genes resulting in altered biomass, root development, and 15NO3- uptake, specifically under low-N conditions. This dynamic N-signaling GRN now provides the temporal "transcriptional logic" for 155 candidate TFs to improve nitrogen use efficiency with potential agricultural applications. Broadly, these time-based approaches can uncover the temporal transcriptional logic for any biological response system in biology, agriculture, or medicine.

A pigeonpea gene confers resistance to Asian soybean rust in soybean.

Asian soybean rust (ASR), caused by the fungus Phakopsora pachyrhizi, is one of the most economically important crop diseases, but is only treatable with fungicides, which are becoming less effective owing to the emergence of fungicide resistance. There are no commercial soybean cultivars with durable resistance to P. pachyrhizi, and although soybean resistance loci have been mapped, no resistance genes have been cloned. We report the cloning of a P. pachyrhizi resistance gene CcRpp1 (Cajanus cajan Resistance against Phakopsora pachyrhizi 1) from pigeonpea (Cajanus cajan) and show that CcRpp1 confers full resistance to P. pachyrhizi in soybean. Our findings show that legume species related to soybean such as pigeonpea, cowpea, common bean and others could provide a valuable and diverse pool of resistance traits for crop improvement.

Genotypic and chemotypic diversity of Neotyphodium endophytes in tall fescue from Greece.

Epichloid endophytes provide protection from a variety of biotic and abiotic stresses for cool-season grasses, including tall fescue. A collection of 85 tall fescue lines from 15 locations in Greece, including both Continental and Mediterranean germplasm, was screened for the presence of native endophytes. A total of 37 endophyte-infected lines from 10 locations were identified, and the endophytes were classified into five distinct groups (G1 to G5) based on physical characteristics such as colony morphology, growth rate, and conidial morphology. These classifications were supported by phylogenetic analyses of housekeeping genes tefA and tubB, and the endophytes were further categorized as Neotyphodium coenophialum isolates (G1, G4, and G5) or Neotyphodium sp. FaTG-2 (Festuca arundinacea taxonomic group 2 isolates (G2 and G3). Analyses of the tall fescue matK chloroplast genes indicated a population-wide, host-specific association between N. coenophialum and Continental tall fescue and between FaTG-2 and Mediterranean tall fescue that was also reflected by differences in colonization of host tillers by the native endophytes. Genotypic analyses of alkaloid gene loci combined with chemotypic (chemical phenotype) profiles provided insight into the genetic basis of chemotype diversity. Variation in alkaloid gene content, specifically the presence and absence of genes, and copy number of gene clusters explained the alkaloid diversity observed in the endophyte-infected tall fescue, with one exception. The results from this study provide insight into endophyte germplasm diversity present in living tall fescue populations.

Epichloe canadensis, a new interspecific epichloid hybrid symbiotic with Canada wildrye (Elymus canadensis).

Many Epichloë endophytes found in cool-season grasses are interspecific hybrids possessing much or all of the genomes of two or three progenitors. Here we characterize Epichloë canadensis sp. nov., a hybrid species inhabiting the grass species Elymus canadensis native to North America. Three distinct morphotypes were identified that were separated into two groups by molecular phylogenetic analysis. Sequence analysis of the translation elongation factor 1-α (tefA) and ß-tubulin (tubB) genes revealed two copies in all isolates examined. Phylogenetic analyses indicated that allele 1 of each gene was derived from Epichloë amarillans and allele 2 from Epichloë elymi. This is the first documentation of an interspecific hybrid endophyte derived from parents of strictly North American origins. Alkaloid gene profiling using primers specific to genes in the peramine, loline, indole-diterpene and ergot alkaloid pathways may indicate chemotypic variation in the ergot alkaloid and loline pathways between the assigned morphotypes. All isolates have the gene enabling the production of peramine but lack genes in the indole-diterpene biosynthesis pathway. Morphology and phylogenetic evidence support the designation of isolates from El. canadensis as a new interspecific hybrid species.

Loss of abaxial leaf epicuticular wax in Medicago truncatula irg1/palm1 mutants results in reduced spore differentiation of anthracnose and nonhost rust pathogens.

To identify genes that confer nonhost resistance to biotrophic fungal pathogens, we did a forward-genetics screen using Medicago truncatula Tnt1 retrotransposon insertion lines. From this screen, we identified an inhibitor of rust germ tube differentation1 (irg1) mutant that failed to promote preinfection structure differentiation of two rust pathogens, Phakopsora pachyrhizi and Puccinia emaculata, and one anthracnose pathogen, Colletotrichum trifolii, on the abaxial leaf surface. Cytological and chemical analyses revealed that the inhibition of rust preinfection structures in irg1 mutants is due to complete loss of the abaxial epicuticular wax crystals and reduced surface hydrophobicity. The composition of waxes on abaxial leaf surface of irg1 mutants had >90% reduction of C30 primary alcohols and a preferential increase of C29 and C31 alkanes compared with the wild type. IRG1 encodes a Cys(2)His(2) zinc finger transcription factor, PALM1, which also controls dissected leaf morphology in M. truncatula. Transcriptome analysis of irg1/palm1 mutants revealed downregulation of eceriferum4, an enzyme implicated in primary alcohol biosynthesis, and MYB96, a major transcription factor that regulates wax biosynthesis. Our results demonstrate that PALM1 plays a role in regulating epicuticular wax metabolism and transport and that epicuticular wax influences spore differentiation of host and nonhost fungal pathogens.

Abundant degenerate miniature inverted-repeat transposable elements in genomes of epichloid fungal endophytes of grasses.

Miniature inverted-repeat transposable elements (MITEs) are abundant repeat elements in plant and animal genomes; however, there are few analyses of these elements in fungal genomes. Analysis of the draft genome sequence of the fungal endophyte Epichloë festucae revealed 13 MITE families that make up almost 1% of the E. festucae genome, and relics of putative autonomous parent elements were identified for three families. Sequence and DNA hybridization analyses suggest that at least some of the MITEs identified in the study were active early in the evolution of Epichloë but are not found in closely related genera. Analysis of MITE integration sites showed that these elements have a moderate integration site preference for 5' genic regions of the E. festucae genome and are particularly enriched near genes for secondary metabolism. Copies of the EFT-3m/Toru element appear to have mediated recombination events that may have abolished synthesis of two fungal alkaloids in different epichloae. This work provides insight into the potential impact of MITEs on epichloae evolution and provides a foundation for analysis in other fungal genomes.