United States Patent Application Publication: COUNTER SELECTION STRATEGY FOR GRAM-NEGATIVE BACTERIA

A Gram-negative bacterium useful for genetically engineering plants is provided. The Gram-negative bacterium contains, as part of genome, an inducible regulatory sequence operatively linked to a nucleotide sequence encoding a levansucrase. Alternatively, the Gram-negative bacterium comprises a recombinant nucleic acid construct containing an inducible regulatory sequence operatively linked to a nucleotide sequence encoding a levansucrase. Also provided are recombinant nucleic acid constructs comprising an inducible regulatory sequence operatively coupled to a nucleotide sequence encoding a levansucrase and a method for transforming plants using the Gram-negative bacterium of the present invention

\u3ci\u3ePhenoImage\u3c/i\u3e: An open-source graphical user interface for plant image analysis

High-throughput genotyping coupled with molecular breeding approaches have dramatically accelerated crop improvement programs. More recently, improved plant phenotyping methods have led to a shift from manual measurements to automated platforms with increased scalability and resolution. Considerable effort has also gone into developing large-scale downstream processing of the imaging datasets derived from high-throughput phenotyping (HTP) platforms. However, most available tools require some programming skills.We developed PhenoImage, an open-source graphical user interface (GUI) based cross-platform solution for HTP image processing intending to make image analysis accessible to users with either little or no programming skills. The open-source nature provides the possibility to extend its usability to meet user-specific requirements. The availability of multiple functions and filtering parameters provides flexibility to analyze images from a wide variety of plant species and platforms. PhenoImage can be run on a personal computer as well as on high-performance computing clusters. To test the efficacy of the application, we analyzed the LemnaTec Imaging system derived red, green, and blue (RGB) color intensity and plant pigmentation-based fluorescence shoot images from two plant species: sorghum [Sorghum bicolor (L.) Moench] and wheat (Triticum aestivum L.) differing in their physical attributes. In the study, we discuss the development, implementation, and working of the PhenoImage

PI‑Plat: a high‑resolution image‑based 3D reconstruction method to estimate growth dynamics of rice inflorescence traits

Background: Recent advances in image-based plant phenotyping have improved our capability to study vegetative stage growth dynamics. However, more complex agronomic traits such as inflorescence architecture (IA), which predominantly contributes to grain crop yield are more challenging to quantify and hence are relatively less explored. Previous efforts to estimate inflorescence-related traits using image-based phenotyping have been limited to destructive end-point measurements. Development of non-destructive inflorescence phenotyping platforms could accelerate the discovery of the phenotypic variation with respect to inflorescence dynamics and mapping of the underlying genes regulating critical yield components. Results: The major objective of this study is to evaluate post-fertilization development and growth dynamics of inflorescence at high spatial and temporal resolution in rice. For this, we developed the Panicle Imaging Platform (PI-Plat) to comprehend multi-dimensional features of IA in a non-destructive manner. We used 11 rice genotypes to capture multi-view images of primary panicle on weekly basis after the fertilization. These images were used to reconstruct a 3D point cloud of the panicle, which enabled us to extract digital traits such as voxel count and color intensity. We found that the voxel count of developing panicles is positively correlated with seed number and weight at maturity. The voxel count from developing panicles projected overall volumes that increased during the grain filling phase, wherein quantification of color intensity estimated the rate of panicle maturation. Our 3D based phenotyping solution showed superior performance compared to conventional 2D based approaches. Conclusions: For harnessing the potential of the existing genetic resources, we need a comprehensive understanding of the genotype-to-phenotype relationship. Relatively low-cost sequencing platforms have facilitated high-throughput genotyping, while phenotyping, especially for complex traits, has posed major challenges for crop improvement. PI-Plat offers a low cost and high-resolution platform to phenotype inflorescence-related traits using 3D reconstruction-based approach. Further, the non-destructive nature of the platform facilitates analyses of the same panicle at multiple developmental time points, which can be utilized to explore the genetic variation for dynamic inflorescence traits in cereals

Inactivation of the entire Arabidopsis group II GH3s confers tolerance to salinity and water deficit

Indole-3-acetic acid (IAA) controls a plethora of developmental processes. Thus, regulation of its concentration is of great relevance for plant performance. Cellular IAA concentration depends on its transport, biosynthesis and the various pathways for IAA inactivation, including oxidation and conjugation. Group II members of the GRETCHEN HAGEN 3 (GH3) gene family code for acyl acid amido synthetases catalysing the conjugation of IAA to amino acids. However, the high degree of functional redundancy among them has hampered thorough analysis of their roles in plant development. In this work, we generated an Arabidopsis gh3.1,2,3,4,5,6,9,17 (gh3oct) mutant to knock out the group II GH3 pathway. The gh3oct plants had an elaborated root architecture, showed an increased tolerance to different osmotic stresses, including an IAA-dependent tolerance to salinity, and were more tolerant to water deficit. Indole-3-acetic acid metabolite quantification in gh3oct plants suggested the existence of additional GH3-like enzymes in IAA metabolism. Moreover, our data suggested that 2-oxindole-3-acetic acid production depends, at least in part, on the GH3 pathway. Targeted stress-hormone analysis further suggested involvement of abscisic acid in the differential response to salinity of gh3oct plants. Taken together, our data provide new insights into the roles of group II GH3s in IAA metabolism and hormone-regulated plant development

The Clubroot Pathogen (\u3ci\u3ePlasmodiophora brassicae\u3c/i\u3e) Influences Auxin Signaling to Regulate Auxin Homeostasis in Arabidopsis

The clubroot disease, caused by the obligate biotrophic protist Plasmodiophora brassicae, affects cruciferous crops worldwide. It is characterized by root swellings as symptoms, which are dependent on the alteration of auxin and cytokinin metabolism. Here, we describe that two different classes of auxin receptors, the TIR family and the auxin binding protein 1 (ABP1) in Arabidopsis thaliana are transcriptionally upregulated upon gall formation. Mutations in the TIR family resulted in more susceptible reactions to the root pathogen. As target genes for the different pathways we have investigated the transcriptional regulation of selected transcriptional repressors (Aux/IAA) and transcription factors (ARF). As the TIR pathway controls auxin homeostasis via the upregulation of some auxin conjugate synthetases (GH3), the expression of selected GH3 genes was also investigated, showing in most cases upregulation. A double gh3 mutant showed also slightly higher susceptibility to P. brassicae infection, while all tested single mutants did not show any alteration in the clubroot phenotype. As targets for the ABP1-induced cell elongation the effect of potassium channel blockers on clubroot formation was investigated. Treatment with tetraethylammonium (TEA) resulted in less severe clubroot symptoms. This research provides evidence for the involvement of two auxin signaling pathways in Arabidopsis needed for the establishment of the root galls by P. brassicae

The Clubroot Pathogen (\u3ci\u3ePlasmodiophora brassicae\u3c/i\u3e) Influences Auxin Signaling to Regulate Auxin Homeostasis in Arabidopsis

The clubroot disease, caused by the obligate biotrophic protist Plasmodiophora brassicae, affects cruciferous crops worldwide. It is characterized by root swellings as symptoms, which are dependent on the alteration of auxin and cytokinin metabolism. Here, we describe that two different classes of auxin receptors, the TIR family and the auxin binding protein 1 (ABP1) in Arabidopsis thaliana are transcriptionally upregulated upon gall formation. Mutations in the TIR family resulted in more susceptible reactions to the root pathogen. As target genes for the different pathways we have investigated the transcriptional regulation of selected transcriptional repressors (Aux/IAA) and transcription factors (ARF). As the TIR pathway controls auxin homeostasis via the upregulation of some auxin conjugate synthetases (GH3), the expression of selected GH3 genes was also investigated, showing in most cases upregulation. A double gh3 mutant showed also slightly higher susceptibility to P. brassicae infection, while all tested single mutants did not show any alteration in the clubroot phenotype. As targets for the ABP1-induced cell elongation the effect of potassium channel blockers on clubroot formation was investigated. Treatment with tetraethylammonium (TEA) resulted in less severe clubroot symptoms. This research provides evidence for the involvement of two auxin signaling pathways in Arabidopsis needed for the establishment of the root galls by P. brassicae

A non-destructive approach for measuring rice panicle-level photosynthetic responses using 3D-image reconstruction

Our understanding of the physiological response of rice inflorescence (panicle) to environmental stresses is limited by the challenge of accurately determining panicle photosynthetic parameters and their impact on grain yield. This is primarily due to lack of a suitable gas exchange methodology for panicles, as well as non-destructive methods to accurately determine panicle surface area. To address these challenges, we have developed a custom panicle gas exchange cylinder compatible with the LiCor 6800 Infra-red Gas Analyzer. Accurate surface area measurements were determined with a 3D panicle imaging platform to normalize the panicle-level photosynthetic measurements. We observed differential responses in both panicle and flag leaf for two temperate Japonica rice genotypes (accessions, TEJ-1 and TEJ-2) exposed to heat stress during early grain filling. There was a notable divergence in relative photosynthetic contribution of flag leaf and panicles for the genotype tolerant to heat stress (TEJ-2) compared to the less tolerant accession. The novelty of this approach is that it is non-destructive and more accurately determines panicle area and photosynthetic parameters, enabling researchers to monitor temporal changes in panicle physiology during the reproductive development. The method is useful for panicle-level measurements under diverse environmental stresses, and for evaluating genotypic variation for panicle physiology and architecture in other cereals with compact inflorescences

Allelic variation in rice \u3ci\u3eFertilization Independent Endosperm 1\u3c/i\u3e contributes to grain width under high night temperature stress

A higher minimum (night-time) temperature is considered a greater limiting factor for reduced rice yield than a similar increase in maximum (daytime) temperature. While the physiological impact of high night temperature (HNT) has been studied, the genetic and molecular basis of HNT stress response remains unexplored. We examined the phenotypic variation for mature grain size (length and width) in a diverse set of rice accessions under HNT stress. Genome-wide association analysis identified several HNT-specific loci regulating grain size as well as loci that are common for optimal and HNT stress conditions. A novel locus contributing to grain width under HNT conditions colocalized with Fie1, a component of the FIS-PRC2 complex. Our results suggest that the allelic difference controlling grain width under HNT is a result of differential transcript-level response of Fie1 in grains developing under HNT stress. We present evidence to support the role of Fie1 in grain size regulation by testing overexpression (OE) and knockout mutants under heat stress. The OE mutants were either unaltered or had a positive impact on mature grain size under HNT, while the knockouts exhibited significant grain size reduction under these conditions

Rice \u3ci\u3eChalky\u3c/i\u3e Grain 5 regulates natural variation for grain quality under heat stress

Heat stress occurring during rice (Oryza sativa) grain development reduces grain quality, which often manifests as increased grain chalkiness. Although the impact of heat stress on grain yield is well-studied, the genetic basis of rice grain quality under heat stress is less explored as quantifying grain quality is less tractable than grain yield. To address this, we used an image-based colorimetric assay (Red, R; and Green, G) for genome-wide association analysis to identify genetic loci underlying the phenotypic variation in rice grains exposed to heat stress. We found the R to G pixel ratio (RG) derived from mature grain images to be effective in distinguishing chalky grains from translucent grains derived from control (28/24°C) and heat stressed (36/32°C) plants. Our analysis yielded a novel gene, rice Chalky Grain 5 (OsCG5) that regulates natural variation for grain chalkiness under heat stress. OsCG5 encodes a grain-specific, expressed protein of unknown function. Accessions with lower transcript abundance of OsCG5 exhibit higher chalkiness, which correlates with higher RG values under stress. These findings are supported by increased chalkiness of OsCG5 knock-out (KO) mutants relative to wildtype (WT) under heat stress. Grains from plants overexpressing OsCG5 are less chalky than KOs but comparable to WT under heat stress. Compared to WT and OE, KO mutants exhibit greater heat sensitivity for grain size and weight relative to controls. Collectively, these results show that the natural variation at OsCG5 may contribute towards rice grain quality under heat stress