Direct and indirect targets of the arabidopsis seed transcription factor ABSCISIC ACID INSENSITIVE3.

Arabidopsis thaliana ABSCISIC ACID INSENSITIVE3 (ABI3) is a transcription factor in the B3 domain family. ABI3, along with B3 domain transcription factors LEAFY COTYLEDON2 (LEC2) and FUSCA3 (FUS3), and LEC1, a subunit of the CCAAT box-binding complex, form the so-called LAFL network to control various aspects of seed development and maturation. ABI3 also contributes to the abscisic acid (ABA) response. We report on chromatin immunoprecipitation-tiling array experiments to map binding sites for ABI3 globally. We also assessed transcriptomes in response to ABI3 by comparing developing abi3-5 and wild-type seeds and combined this information to ascertain direct and indirect responsive ABI3 target genes. ABI3 can induce and repress its transcription of target genes directly and some intriguing differences exist in cis motifs between these groups of genes. Directly regulated targets reflect the role of ABI3 in seed maturation, desiccation tolerance, entry into a quiescent state and longevity. Interestingly, ABI3 directly represses a gene encoding a microRNA (MIR160B) that targets AUXIN RESPONSE FACTOR (ARF)10 and ARF16 that are involved in establishment of dormancy. In addition, ABI3, like FUS3, regulates genes encoding MIR156 but while FUS3 only induces genes encoding this product, ABI3 induces these genes during the early stages of seed development, but represses these genes during late development. The interplay between ABI3, the other LAFL genes, and the VP1/ABI3-LIKE (VAL) genes, which are involved in the transition to seedling development are examined and reveal complex interactions controlling development.

Draft Genome Sequence Resource for the Orange Rust Pathogen of Sugarcane Puccinia kuehnii.

Puccinia kuehnii is an obligate biotrophic fungal pathogen that causes orange rust of sugarcane, which is prevalent in many countries around the globe. In the United States, orange rust was first detected in sugarcane in Florida in 2007 and poses a persistent and economically damaging threat to the sugarcane industry in this region. Here, we generated the first genome assemblies for two isolates of P. kuehnii (1040 and 2143) collected in Florida in 2017 from two sugarcane cultivars, CL85-1040 and CP89-2143, respectively. These two rust genome resources will be of immense value for future genomic studies, particularly further exploration of the predicted secretomes that may help define key pathogenicity determinants for this economically important pathogen.

Draft Genome Sequence Resource of the Citrus Stem-End Rot Fungal Pathogen Lasiodiplodia theobromae CITRA15.

Lasiodiplodia theobromae is a fungal pathogen associated with perennial tropical fruit plants worldwide. In citrus, L. theobromae causes stem-end rot (Diplodia stem-end rot), a damaging postharvest disease that is aggravated when trees are also infected with the citrus greening bacteria 'Candidatus Liberibacter asiaticus'. Due to the latent infection of L. theobromae during the preharvest stage, it becomes difficult to control the disease by chemical or physical treatment. In the current study, we sequenced and assembled strain CITRA15, the first genome of L. theobromae obtained from diseased Citrus paradise 'Flame' grapefruit in Florida, and thereby provided a genomic resource for future research on diagnostics, and postharvest and preharvest disease management of citrus and other fruit crops.

Isoflavone levels, nodulation and gene expression profiles of a CRISPR/Cas9 deletion mutant in the isoflavone synthase gene of red clover.

KEY MESSAGE: Isoflavones are not involved in rhizobial signaling in red clover, but likely play a role in defense in the rhizosphere. Red clover (Trifolium pratense) is a high-quality forage legume, well suited for grazing and hay production in the temperate regions of the world. Like many legumes, red clover produces a number of phenylpropanoid compounds including anthocyanidins, flavan-3-ols, flavanols, flavanones, flavones, and isoflavones. The study of isoflavone biosynthesis and accumulation in legumes has come into the forefront of biomedical and agricultural research due to potential for medicinal, antimicrobial, and environmental implications. CRISPR/Cas9 was used to knock out the function of a key enzyme in the biosynthesis of isoflavones, isoflavone synthase (IFS1). A hemizygous plant carrying a 9-bp deletion in the IFS1 gene was recovered and was intercrossed to obtain homozygous mutant plants. Levels of the isoflavones formononetin, biochanin A and genistein were significantly reduced in the mutant plants. Wild-type and mutant plants were inoculated with rhizobia to test the effect of the mutation on nodulation, but no significant differences were observed, suggesting that these isoflavones do not play important roles in nodulation. Gene expression profiling revealed an increase in expression of the upstream genes producing the precursors for IFS1, namely, phenylalanine ammonium lyase and chalcone synthase, but there were no significant differences in IFS1 gene expression or in the downstream genes in the production of specific isoflavones. Higher expression in genes involved in ethylene response was observed in the mutant plants. This response is normally associated with biotic stress, suggesting that the plants may have been responding to cues in the surrounding rhizosphere due to lower levels of isoflavones.

Microsymbiont discrimination mediated by a host-secreted peptide in Medicago truncatula.

The legume-rhizobial symbiosis results in the formation of root nodules that provide an ecological niche for nitrogen-fixing bacteria. However, plant-bacteria genotypic interactions can lead to wide variation in nitrogen fixation efficiency, and it is not uncommon that a bacterial strain forms functional (Fix+) nodules on one plant genotype but nonfunctional (Fix-) nodules on another. Host genetic control of this specificity is unknown. We herein report the cloning of the Medicago truncatula NFS1 gene that regulates the fixation-level incompatibility with the microsymbiont Sinorhizobium meliloti Rm41. We show that NFS1 encodes a nodule-specific cysteine-rich (NCR) peptide. In contrast to the known role of NCR peptides as effectors of endosymbionts' differentiation to nitrogen-fixing bacteroids, we demonstrate that specific NCRs control discrimination against incompatible microsymbionts. NFS1 provokes bacterial cell death and early nodule senescence in an allele-specific and rhizobial strain-specific manner, and its function is dependent on host genetic background.

Gene Regulation by the AGL15 Transcription Factor Reveals Hormone Interactions in Somatic Embryogenesis.

The MADS box transcription factor Arabidopsis (Arabidopsis thaliana) AGAMOUS-LIKE15 (AGL15) and a putative ortholog from soybean (Glycine max), GmAGL15, are able to promote somatic embryogenesis (SE) in these plants when ectopically expressed. SE is an important means of plant regeneration, but many plants, or even particular cultivars, are recalcitrant for this process. Understanding how (Gm)AGL15 promotes SE by identifying and characterizing direct and indirect downstream regulated genes can provide means to improve regeneration by SE for crop improvement and to perform molecular tests of genes. Conserved transcription factors and the genes they regulate in common between species may provide the most promising avenue to identify targets for SE improvement. We show that (Gm)AGL15 negatively regulates auxin signaling in both Arabidopsis and soybean at many levels of the pathway, including the repression of AUXIN RESPONSE FACTOR6 (ARF6) and ARF8 and TRANSPORT INHIBITOR RESPONSE1 as well as the indirect control of components via direct expression of a microRNA-encoding gene. We demonstrate interaction between auxin and gibberellic acid in the promotion of SE and document an inverse correlation between bioactive gibberellic acid and SE in soybean, a difficult crop to transform. Finally, we relate hormone accumulation to transcript accumulation of important soybean embryo regulatory factors such as ABSCISIC ACID INSENSITIVE3 and FUSCA3 and provide a working model of hormone and transcription factor interaction in the control of SE.

Alterations in the transcriptome of soybean in response to enhanced somatic embryogenesis promoted by orthologs of Agamous-like15 and Agamous-like18.

Somatic embryogenesis (SE) is a poorly understood process during which competent cells respond to inducing conditions, allowing the development of somatic embryos. It is important for the regeneration of transgenic plants, including for soybean (Glycine max). We report here that constitutive expression of soybean orthologs of the Arabidopsis (Arabidopsis thaliana) MADS box genes Agamous-like15 (GmAGL15) and GmAGL18 increased embryogenic competence of explants from these transgenic soybean plants. To understand how GmAGL15 promotes SE, expression studies were performed. Particular genes of interest involved in embryogenesis (abscisic acid-insensitive3 and FUSCA3) were found to be directly up-regulated by GmAGL15 by using a combination of quantitative reverse transcription-polymerase chain reaction and chromatin immunoprecipitation. To look more broadly at changes in gene expression in response to GmAGL15, we assessed the transcriptome using the Affymetrix Soybean Genome Array. Interestingly, the gene expression profile of 35Spro:GmAGL15 explants (0 d in culture) was found to resemble nontransgenic tissue that had been induced for SE by being placed on induction medium for 3 d, possibly explaining the more rapid SE development observed on 35Spro:GmAGL15 tissue. In particular, transcripts from genes related to the stress response showed increased transcript accumulation in explants from 35Spro:GmAGL15 tissue. These same genes also showed increased transcript accumulation in response to culturing nontransgenic soybean explants on the medium used to induce SE. Overexpression of GmAGL15 may enhance SE by making the tissue more competent to respond to 2,4-dichlorophenoxyacetic acid induction by differential regulation of genes such as those involved in the stress response, resulting in more rapid and prolific SE.

Identification of a dominant gene in Medicago truncatula that restricts nodulation by Sinorhizobium meliloti strain Rm41.

BACKGROUND: Leguminous plants are able to form a root nodule symbiosis with nitrogen-fixing soil bacteria called rhizobia. This symbiotic association shows a high level of specificity. Beyond the specificity for the legume family, individual legume species/genotypes can only interact with certain restricted group of bacterial species or strains. Specificity in this system is regulated by complex signal exchange between the two symbiotic partners and thus multiple genetic mechanisms could be involved in the recognition process. Knowledge of the molecular mechanisms controlling symbiotic specificity could enable genetic improvement of legume nitrogen fixation, and may also reveal the possible mechanisms that restrict root nodule symbiosis in non-legumes. RESULTS: We screened a core collection of Medicago truncatula genotypes with several strains of Sinorhizobium meliloti and identified a naturally occurring dominant gene that restricts nodulation by S. meliloti Rm41. We named this gene as Mt-NS1 (for M.truncatulanodulation specificity 1). We have mapped the Mt-NS1 locus within a small genomic region on M. truncatula chromosome 8. The data reported here will facilitate positional cloning of the Mt-NS1 gene. CONCLUSIONS: Evolution of symbiosis specificity involves both rhizobial and host genes. From the bacterial side, specificity determinants include Nod factors, surface polysaccharides, and secreted proteins. However, we know relatively less from the host side. We recently demonstrated that a component of this specificity in soybeans is defined by plant NBS-LRR resistance (R) genes that recognize effector proteins delivered by the type III secretion system (T3SS) of the rhizobial symbionts. However, the lack of a T3SS in many sequenced S. meliloti strains raises the question of how the specificity is regulated in the Medicago-Sinorhizobium system beyond Nod-factor perception. Thus, cloning and characterization of Mt-NS1 will add a new dimension to our knowledge about the genetic control of nodulation specificity in the legume-rhizobial symbiosis.

AGAMOUS-Like15 promotes somatic embryogenesis in Arabidopsis and soybean in part by the control of ethylene biosynthesis and response.

Many of the regulatory processes occurring during plant embryogenesis are still unknown. Relatively few cells are involved, and they are embedded within maternal tissues, making this developmental phase difficult to study. Somatic embryogenesis is a more accessible system, and many important regulatory genes appear to function similar to zygotic development, making somatic embryogenesis a valuable model for the study of zygotic processes. To better understand the role of the Arabidopsis (Arabidopsis thaliana) MADS factor AGAMOUS-Like15 (AGL15) in the promotion of somatic embryogenesis, direct target genes were identified by chromatin immunoprecipitation-tiling arrays and expression arrays. One potential directly up-regulated target was At5g61590, which encodes a member of the ethylene response factor subfamily B-3 of APETALA2/ethylene response factor transcription factors and is related to Medicago truncatula somatic embryo-related factor1 (MtSERF1), which has been shown to be required for somatic embryogenesis in M. truncatula. Here, we report confirmation that At5g61590 is a directly expressed target of AGL15 and that At5g61590 is essential for AGL15's promotion of somatic embryogenesis. Because At5g61590 is a member of the ethylene response factor family, effects of ethylene on somatic embryogenesis were investigated. Precursors to ethylene stimulate somatic embryogenesis, whereas inhibitors of ethylene synthesis or perception reduce somatic embryogenesis. To extend findings to a crop plant, we investigated the effects of ethylene on somatic embryogenesis in soybean (Glycine max). Furthermore, we found that a potential ortholog of AGL15 in soybean (GmAGL15) up-regulates ethylene biosynthesis and response, including direct regulation of soybean orthologs of At5g61590/MtSERF1 named here GmSERF1 and GmSERF2, in concordance with the M. truncatula nomenclature.

Identification of QTLs Conferring Resistance to Bacterial Diseases in Rice.

Bacterial panicle blight, bacterial leaf streak, and bacterial brown stripe are common bacterial diseases in rice that represent global threats to stable rice yields. In this study, we used the rice variety HZ, Nekken and their 120 RIL population as experimental materials. Phenotypes of the parents and RILs were quantitatively analyzed after inoculation with Burkholderia glumae, Xanthomonas oryzae pv. oryzicola, and Acidovorax avenae subsp. avenae. Genetic SNP maps were also constructed and used for QTL mapping of the quantitative traits. We located 40 QTL loci on 12 chromosomes. The analysis of disease resistance-related candidate genes in the QTL regions with high LOD value on chromosomes 1, 3, 4, and 12 revealed differential expression before and after treatment, suggesting that the identified genes mediated the variable disease resistance profiles of Huazhan and Nekken2. These results provide an important foundation for cloning bacterial-resistant QTLs of panicle blight, leaf streak, and brown stripe in rice.

Involvement of negative feedback regulation in wound-induced ethylene synthesis in 'Saijo' persimmon.

Wounding is one of the most effective stress signals to induce ethylene synthesis in persimmon (Diospyros kaki Thunb.). We found that wound-induced ethylene biosynthesis is subjected to negative feedback regulation in mature 'Saijo' persimmon fruit since ethylene production was enhanced by 1-methylcyclopropene (1-MCP) (an inhibitor of ethylene perception) pretreatment, which was approximately 1.8 fold of that in control tissues (without 1-MCP pretreatment). Wound-induced 1-aminocyclopropane-1-carboxylate (ACC) synthase activity and DK-ACS2 gene expression were substantially increased by 1-MCP pretreatment after 12 h, which resulted in much higher ACC content in 1-MCP pretreated tissues than that in a control after 24 h. These results indicated that wound-induced DK-ACS2 gene expression was negatively regulated by ethylene in mature persimmon fruit. However, 1-MCP pretreatment had no effect on DK-ACO1 gene expression, suggesting the independence of wound-induced DK-ACO1 on ethylene. Out of accord with DK-ACO1 gene expression, ACC oxidase activity was enhanced 48 h after wounding in 1-MCP pretreated tissues, reaching a peak 1.5-fold higher than that in control tissues at 60 h.

Transcriptome analysis indicates that GmAGAMOUS-Like 15 may enhance somatic embryogenesis by promoting a dedifferentiated state.

Somatic embryogenesis (SE) is an important avenue for regeneration of many plants. Although documented over half a century ago, the process of SE remains poorly understood and many factors impact upon competence for SE. We recently reported that a Glycine max ortholog of a MADS-domain transcription factor that promotes SE in Arabidopsis also enhances SE in soybean. We recently assessed transcriptomes in 35Spro:GmAGL15 compared to control during an early time-course of SE and in response to 35Spro:AtAGL15. We expand here upon discussion of the types of genes regulated by overexpression of AGL15 and characterize the step of SE that may be affected by altered accumulation of AGL15.

Decreased GmAGL15 expression and reduced ethylene synthesis may contribute to reduced somatic embryogenesis in a poorly embryogenic cultivar of Glycine max.

Somatic embryogenesis (SE) is the process by which cells become dedifferentiated and reprogram to follow an embryogenic pathway. It is important for regeneration of transgenic plants as well as for propagation of certain genotypes. However, competence for SE varies, even among genotypes of a species, and the basis for this variation is not understood. We have found that the MADS-box transcription factor (Glycine max) AGAMOUS-Like 15 [(Gm)AGL15] promotes SE in Arabidopsis and in soybean when overexpressed. In soybean, part of the promotion of SE is via GmAGL15-mediated control of ethylene biosynthesis and response. Addition of ACC, the precursor to ethylene, to culture media enhanced SE in Arabidopsis and soybean. Transcription factors important for embryogenesis responded directly to GmAGL15 and to ethylene accumulation. Here we correlate ethylene production and patterns of gene expression with SE potential of soybean genotypes. However, other results indicate that there is not a complete positive correlation between ethylene production and SE, indicating that the interactions between hormones, gene expression and developmental outcomes are complex.