This manuscript provides genome-level analysis of disease resistance genes in four maize lines, including studies of haplotype and resistance gene number as well as selection and recombination. The Rp1 locus of maize is a complex resistance gene (R-gene) cluster that confers race-specific resistance to Puccinia sorghi, the causal agent of common leaf rust. Rp1 NB-LRR disease resistance genes were isolated from two Rp1 haplotypes (HRp1-B and HRp1-M) and two maize inbred lines (B73 and H95). Sixty-one Rp1 genes were isolated from Rp1-B, Rp1-M, B73 and H95 with a PCR-based approach. The four maize lines carried from 12 to 19 Rp1 genes. From 4 to 9 of the identified Rp1 genes were transcribed in the four maize lines. The Rp1 gene nucleotide diversity was higher in HRp1-B and HRp1-M than in B73 and H95. Phylogenic analysis of 69 Rp1 genes revealed that the Rp1 genes maintained in HRp1-B, HRp1-M and H95 are evolving independently of each other, while Rp1 genes in B73 and HRp1-D appear more like each other than they do genes in the other lines. The results also revealed that the analysed Rp1 R-genes were under positive selection in HRp1-M and B73. Intragenic recombination was detected in Rp1 genes maintained in the four maize lines. This demonstrates that a genetic process that has the potential to generate new resistance genes with new specificities is active at the Rp1 locus in the four analysed maize lines and that the new resistance genes may act against newly arising pathogen races that become prevalent in the pathogen population.

The protozoan parasite responsible for human amoebiasis is Entamoeba histolytica. An important facet of the life cycle of E. histolytica involves the conversion of the mature trophozoite to a cyst. This transition is thought to involve homologous recombination (HR), which is dependent upon the Rad51 recombinase. Here, a biochemical characterization of highly purified ehRad51 protein is presented. The ehRad51 protein preferentially binds ssDNA, forms a presynaptic filament and possesses ATP hydrolysis activity that is stimulated by the presence of DNA. Evidence is provided that ehRad51 catalyzes robust DNA strand exchange over at least 5.4 kilobase pairs. Although the homologous DNA pairing activity of ehRad51 is weak, it is strongly enhanced by the presence of two HR accessory cofactors, calcium and Hop2-Mnd1. The biochemical system described herein was used to demonstrate the potential for targeting ehRad51 with two small molecule inhibitors of human RAD51. We show that 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) inhibited ehRad51 by interfering with DNA binding and attenuated encystation in Entamoeba invadens, while B02 had no effect on ehRad51 strand exchange activity. These results provide insight into the underlying mechanism of homology-directed DNA repair in E. histolytica.

Nodule number regulation in legumes is controlled by a feedback loop that integrates nutrient and rhizobia symbiont status signals to regulate nodule development. Signals from the roots are perceived by shoot receptors, including a CLV1-like receptor-like kinase known as SUNN in the annual medicMedicago truncatula. In the absence of functional SUNN, the autoregulation feedback loop is disrupted, resulting in hypernodulation. To elucidate early autoregulation mechanisms disrupted in SUNN mutants, we searched for genes with altered expression in the loss-of-functionsunn-4mutant and included therdn1-2autoregulation mutant for comparison. We identified constitutively altered expression of small groups of genes insunn-4roots, including higher levels of transcription factorNF-YA2, and insunn-4shoots. All genes with verified roles in nodulation that were induced in wild type roots during the establishment of nodules were also induced insunn-4, including, surprisingly, autoregulation genesTML2andTML1. Among all genes with a differential response to rhizobia in wild type roots, only an isoflavone-7-O-methyltransferase gene (Medtr7g014510) was found to be unresponsive insunn-4. In shoot tissues of wild type, eight rhizobia-responsive genes were identified, including a MYB family transcription factor gene (Medtr3111880) which remained at a baseline level insunn-4; three genes were found to be induced by rhizobia in shoots ofsunn-4but not wild type. We also cataloged the temporal induction profiles of many small secreted peptide (MtSSP) genes in nodulating root tissues, encompassing members of twenty-four peptide families, including the CLE and IRON MAN families. The discovery that expression ofTMLgenes in roots, a key factor in inhibiting nodulation in response to autoregulation signals, is also triggered insunn-4in the section of roots analyzed suggests that the mechanism ofTMLregulation inM. truncatulamay be more complex than published models.

Root nodulation results from a symbiotic relationship between a plant host and Rhizobium bacteria. Synchronized gene expression patterns over the course of rhizobial infection result in activation of pathways that are unique but overlapping with the highly conserved pathways that enable mycorrhizal symbiosis. We performed RNA sequencing of 30 Medicago truncatula root maturation zone samples at five distinct time points. These samples included plants inoculated with Sinorhizobium medicae and control plants that did not receive any Rhizobium. Following gene expression quantification, we identified 1,758 differentially expressed genes at various time points. We constructed a gene co-expression network (GCN) from the same data and identified link community modules (LCMs) that were comprised entirely of differentially expressed genes at specific time points post-inoculation. One LCM included genes that were up-regulated at 24 h following inoculation, suggesting an activation of allergen family genes and carbohydrate-binding gene products in response to Rhizobium. We also identified two LCMs that were comprised entirely of genes that were down regulated at 24 and 48 h post-inoculation. The identity of the genes in these modules suggest that down-regulating specific genes at 24 h may result in decreased jasmonic acid production with an increase in cytokinin production. At 48 h, coordinated down-regulation of a specific set of genes involved in lipid biosynthesis may play a role in nodulation. We show that GCN-LCM analysis is an effective method to preliminarily identify polygenic candidate biomarkers of root nodulation and develop hypotheses for future discovery.