Pamela Abbruscato

Characterization of WRKY co-regulatory networks in rice and Arabidopsis

BackgroundThe WRKY transcription factor gene family has a very ancient origin and has undergone extensive duplications in the plant kingdom. Several studies have pointed out their involvement in a range of biological processes, revealing that a large number of WRKY genes are transcriptionally regulated under conditions of biotic and/or abiotic stress. To investigate the existence of WRKY co-regulatory networks in plants, a whole gene family WRKYs expression study was carried out in rice (Oryza sativa). This analysis was extended to Arabidopsis thaliana taking advantage of an extensive repository of gene expression data.ResultsThe presented results suggested that 24 members of the rice WRKY gene family (22% of the total) were differentially-regulated in response to at least one of the stress conditions tested. We defined the existence of nine OsWRKY gene clusters comprising both phylogenetically related and unrelated genes that were significantly co-expressed, suggesting that specific sets of WRKY genes might act in co-regulatory networks. This hypothesis was tested by Pearson Correlation Coefficient analysis of the Arabidopsis WRKY gene family in a large set of Affymetrix microarray experiments. AtWRKYs were found to belong to two main co-regulatory networks (COR-A, COR-B) and two smaller ones (COR-C and COR-D), all including genes belonging to distinct phylogenetic groups. The COR-A network contained several AtWRKY genes known to be involved mostly in response to pathogens, whose physical and/or genetic interaction was experimentally proven. We also showed that specific co-regulatory networks were conserved between the two model species by identifying Arabidopsis orthologs of the co-expressed OsWRKY genes.ConclusionIn this work we identified sets of co-expressed WRKY genes in both rice and Arabidopsis that are functionally likely to cooperate in the same signal transduction pathways. We propose that, making use of data from co-regulatory networks, it is possible to highlight novel clusters of plant genes contributing to the same biological processes or signal transduction pathways. Our approach will contribute to unveil gene cooperation pathways not yet identified by classical genetic analyses. This information will open new routes contributing to the dissection of WRKY signal transduction pathways in plants.

Comparative transcriptome profiling of the early response to Magnaporthe oryzae in durable resistant vs susceptible rice (Oryza sativa L.) genotypes.

Durable resistance to blast, the most significant fungal disease of rice, represents an agronomically relevant character. Gigante Vercelli (GV) and Vialone Nano (VN) are two old temperate japonica Italian rice cultivars with contrasting response to blast infection: GV displays durable and broad resistance while VN is highly susceptible. RNA-seq was used to dissect the early molecular processes deployed during the resistance response of GV at 24 h after blast inoculation. Differential gene expression analysis identified 1,070 and 1,484 modulated genes, of which 726 and 699 were up regulated in response to infection in GV and VN, respectively. Gene ontology (GO) enrichment analyses revealed a set of GO terms enriched in both varieties but, despite this commonality, the gene sets contributing to common GO enriched terms were dissimilar. The expression patterns of genes grouped in GV-specific enriched GO terms were examined in detail including at the transcript isoform level. GV exhibited a dramatic up-regulation of genes encoding diterpene phytoalexin biosynthetic enzymes, flavin-containing monooxygenase, class I chitinase and glycosyl hydrolase 17. The sensitivity and high dynamic range of RNA-seq allowed the identification of genes critically involved in conferring GV resistance during the early steps of defence perception-signalling. These included chitin oligosaccharides sensing factors, wall associated kinases, MAPK cascades and WRKY transcription factors. Candidate genes with expression patterns consistent with a potential role as GV-specific functional resistance (R) gene(s) were also identified. This first application of RNA-seq to dissect durable blast resistance supports a crucial role of the prompt induction of a battery of responses including defence-related genes as well as members of gene families involved in signalling and pathogen-related gene expression regulation.

OsWRKY22, a monocot WRKY gene, plays a role in the resistance response to blast

With the aim of identifying novel regulators of host and nonhost resistance to fungi in rice, we carried out a systematic mutant screen of mutagenized lines. Two mutant wrky22 knockout lines revealed clear-cut enhanced susceptibility to both virulent and avirulent Magnaporthe oryzae strains and altered cellular responses to nonhost Magnaporthe grisea and Blumeria graminis fungi. In addition, the analysis of the pathogen responses of 24 overexpressor OsWRKY22 lines revealed enhanced resistance phenotypes on infection with virulent M. oryzae strain, confirming that OsWRKY22 is involved in rice resistance to blast. Bioinformatic analyses determined that the OsWRKY22 gene belongs to a well-defined cluster of monocot-specific WRKYs. The co-regulatory analysis revealed no significant co-regulation of OsWRKY22 with a representative panel of OsWRKYs, supporting its unique role in a series of transcriptional responses. In contrast, inquiring a subset of biotic stress-related Affymetrix data, a large number of resistance and defence-related genes were found to be putatively co-expressed with OsWRKY22. Taken together, all gathered experimental evidence places the monocot-specific OsWRKY22 gene at the convergence point of signal transduction circuits in response to both host and nonhost fungi encountering rice plants.

Assessment of genetic diversity in Italian rice germplasm related to agronomic traits and blast resistance (Magnaportheoryzae)

Italy is the only country in Europe with a significant land area used for rice production. In this paper, the genetic diversity of 172 national varieties and 47 foreign accessions (ITALORYZA collection) was investigated using a set of neutral markers evenly distributed throughout the 12 chromosomes. Out of the 218 alleles detected in our analysis, 17 and 29% were specific to the Italian and foreign accessions, respectively. From the neighbour-joining tree generated, six sub-groups of temperate japonica germplasm were identified. Plant height and grain type measured in the source collection were fitted to the phylogenetic tree, along with the period of variety registration. This integrated genotype–phenotype analysis revealed that specific sub-groups are characterized by uniform classes of grain type, or by similar plant size, or by period of release in the market. The whole collection was also evaluated for leaf blast resistance by inoculating the plants with three strains of Magnaporthe oryzae, representing the pathogen genetic diversity existing in Italy. Only 15 out of 172 Italian accessions (8.7%) were resistant to all three fungal strains. The correlation between genotype and leaf blast phenotype revealed that the most highly resistant Italian varieties are included in a single germplasm sub-group derived from US varieties. This study represents the starting point for carrying out detailed phenotype–genotype whole-genome association studies and identification of the genetic basis of important agronomic traits for rice cultivation in temperate climates.

Genome-Wide Association Study for Traits Related to Plant and Grain Morphology, and Root Architecture in Temperate Rice Accessions

Background In this study we carried out a genome-wide association analysis for plant and grain morphology and root architecture in a unique panel of temperate rice accessions adapted to European pedo-climatic conditions. This is the first study to assess the association of selected phenotypic traits to specific genomic regions in the narrow genetic pool of temperate japonica. A set of 391 rice accessions were GBS-genotyped yielding—after data editing—57000 polymorphic and informative SNPS, among which 54% were in genic regions. Results In total, 42 significant genotype-phenotype associations were detected: 21 for plant morphology traits, 11 for grain quality traits, 10 for root architecture traits. The FDR of detected associations ranged from 3 · 10−7 to 0.92 (median: 0.25). In most cases, the significant detected associations co-localised with QTLs and candidate genes controlling the phenotypic variation of single or multiple traits. The most significant associations were those for flag leaf width on chromosome 4 (FDR = 3 · 10−7) and for plant height on chromosome 6 (FDR = 0.011). Conclusions We demonstrate the effectiveness and resolution of the developed platform for high-throughput phenotyping, genotyping and GWAS in detecting major QTLs for relevant traits in rice. We identified strong associations that may be used for selection in temperate irrigated rice breeding: e.g. associations for flag leaf width, plant height, root volume and length, grain length, grain width and their ratio. Our findings pave the way to successfully exploit the narrow genetic pool of European temperate rice and to pinpoint the most relevant genetic components contributing to the adaptability and high yield of this germplasm. The generated data could be of direct use in genomic-assisted breeding strategies.

CYP72A67 Catalyzes a Key Oxidative Step in Medicago truncatula Hemolytic Saponin Biosynthesis

In the Medicago genus, triterpenic saponins are bioactive secondary metabolites constitutively synthesized in the aerial and subterranean parts of plants via the isoprenoid pathway. Exploitation of saponins as pharmaceutics, agrochemicals and in the food and cosmetic industries has raised interest in identifying the enzymes involved in their synthesis. We have identified a cytochrome P450 (CYP72A67) involved in hemolytic sapogenin biosynthesis by a reverse genetic TILLING approach in a Medicago truncatula ethylmethanesulfonate (EMS) mutagenized collection. Genetic and biochemical analyses, mutant complementation, and expression of the gene in a microsome yeast system showed that CYP72A67 is responsible for hydroxylation at the C-2 position downstream of oleanolic acid synthesis. The affinity of CYP72A67 for substrates with different substitutions at multiple carbon positions was investigated in the same in vitro yeast system, and in relation to two other CYP450s (CYP72A68) responsible for the production of medicagenic acid, the main sapogenin in M. truncatula leaves and roots. Full sib mutant and wild-type plants were compared for their sapogenin profile, expression patterns of the genes involved in sapogenin synthesis, and response to inoculation with Sinorhizobium meliloti. The results obtained allowed us to revise the hemolytic sapogenin pathway in M. truncatula and contribute to highlighting the tissue specificities (leaves/roots) of sapogenin synthesis.

Analysis of transcript and metabolite levels in Italian rice (Oryza sativa L.) cultivars subjected to osmotic stress or benzothiadiazole treatment

One of the major objectives of rice (Oryza sativa L.) breeding programs is the development of new varieties with higher tolerance/resistance to both abiotic and biotic stresses. In this study, Italian rice cultivars were subjected to osmotic stress or benzothiadiazole (BTH) treatments. An analysis of the expression of selected genes known to be involved in the stress response and (1)H nuclear magnetic resonance ((1)H NMR) metabolic profiling were combined with multivariate statistical analyses to elucidate potential correlations between gene expression or metabolite content and observed tolerant/resistant phenotypes. We observed that the expression of three chosen genes (two WRKY genes and one peroxidase encoding gene) differed between susceptible and resistant cultivars in response to BTH treatments. Moreover, the analysis of metabolite content, in particular in the osmotic stress experiment, enabled discrimination between selected cultivars based on differences in the accumulation of some primary metabolites, primarily sugars. This research highlights the potential usefulness of this approach to characterise rice varieties based on transcriptional or metabolic changes due to adverse environmental conditions.

Rice bacterial endophytes: isolation of a collection, identification of beneficial strains and microbiome analysis

Endophytes are harmless or beneficial microorganisms that live inside plants between cells. The relationship they develop with the plant as well as their potential role in plant health is at large unexplored and it is believed that the opportunity to find new and interesting endophytes among the large variety of plants is great. Here, we present the isolation and analysis of a large collection of endophytes from one cultivar of rice grown in Italy. A total 1318 putative endophytes were isolated from roots, leaves and stems from rice grown in submerged and dry conditions and a working collection of 229 isolates was created. Among these, several isolates were confirmed to be endophytes and a few displayed the trait of plant growth promotion. A cultivation independent analysis via 16S rDNA amplicons of the bacterial community of the endosphere was also performed providing information on bacterial diversity in the rice endopshere.

Draft Genome Sequence of Rice Endophyte-Associated Isolate Kosakonia oryzae KO348

ABSTRACT Kosakonia oryzae KO348 is an endophytic and plant growth-promoting strain isolated from the roots of rice in Italy. Here, we report the draft genome sequence of Kosakonia oryzae KO348.

Draft Genome Sequence of the Plant Pathogen Dickeya zeae DZ2Q, Isolated from Rice in Italy

ABSTRACT Dickeya zeae is an emerging rice (Oryza sativa) pathogen causing bacterial foot rot. Related pathogens affect maize (Zea mays) and potato (Solanum tuberosum) and a variety of important ornamental and floral plants. Here, we present the draft genome sequence of D. zeae DZ2Q, an isolate obtained from rice grown in Italy.