Production of yellow-flowered gentian plants by genetic engineering of betaxanthin pigments.

Genetic engineering of flower color provides biotechnological products such as blue carnations or roses by accumulating delphinidin-based anthocyanins not naturally existing in these plant species. Betalains are another class of pigments that in plants are only synthesized in the order Caryophyllales. Although they have been engineered in several plant species, especially red-violet betacyanins, the yellow betaxanthins have yet to be engineered in ornamental plants. We attempted to produce yellow-flowered gentians by genetic engineering of betaxanthin pigments. First, white-flowered gentian lines were produced by knocking out the dihydroflavonol 4-reductase (DFR) gene using CRISPR/Cas9-mediated genome editing. Beta vulgaris BvCYP76AD6 and Mirabilis jalapa MjDOD, driven by gentian petal-specific promoters, flavonoid 3',5'-hydroxylase (F3'5'H) and anthocyanin 5,3'-aromatic acyltransferase (AT), respectively, were transformed into the above DFR-knockout white-flowered line; the resultant gentian plants had vivid yellow flowers. Expression analysis and pigment analysis revealed petal-specific expression and accumulation of seven known betaxanthins in their petals to c. 0.06-0.08 µmol g FW-1 . Genetic engineering of vivid yellow-flowered plants can be achieved by combining genome editing and a suitable expression of betaxanthin-biosynthetic genes in ornamental plants.

Efficient double-flowered gentian plant production using the CRISPR/Cas9 system.

Japanese cultivated gentians are highly valued ornamental flowers in Japan, but the flower shape is mostly limited to the single-flower type, unlike other flowers such as roses and carnations. To overcome this limitation, we used the CRISPR/Cas9 genome editing system to increase double-flowered genetic resources in gentians. Our approach targeted an AGAMOUS (AG) floral homeotic gene (AG1), which is responsible for the natural mutation that causes double flowers in gentians. We designed two targets in exon 1 of AG1 for genome editing and found that 9 of 12 herbicide-resistant shoots had biallelic mutations in the target regions of AG1. These nine lines all produced double flowers, with stamens converted into petaloid organs, similar to the natural mutant. We also analyzed the off-target effects of AG2, which is homologous to AG1, and found that such effects occurred in gentian genome editing but with low frequency. Furthermore, we successfully produced transgene-free genome-edited plants (null segregants) by crossing with wild-type pollen. F1 seedlings were subjected to PCR analysis to determine whether foreign DNA sequences, two partial regions of the CaMV35S promoter and Cas9 gene, were present in the genome. As a result, foreign genes were segregated at a 1 : 1 ratio, indicating successful null segregant production. Using PCR analysis, we confirmed that four representative null segregants did not contain transfer DNA. In summary, our study demonstrates that the CRISPR/Cas9 system can efficiently produce double-flowered gentians, and null segregants can also be obtained. These genome-edited plants are valuable genetic resources for future gentian breeding programs.

A genetically linked pair of NLR immune receptors shows contrasting patterns of evolution.

Throughout their evolution, plant nucleotide-binding leucine-rich-repeat receptors (NLRs) have acquired widely divergent unconventional integrated domains that enhance their ability to detect pathogen effectors. However, the functional dynamics that drive the evolution of NLRs with integrated domains (NLR-IDs) remain poorly understood. Here, we reconstructed the evolutionary history of an NLR locus prone to unconventional domain integration and experimentally tested hypotheses about the evolution of NLR-IDs. We show that the rice (Oryza sativa) NLR Pias recognizes the effector AVR-Pias of the blast fungal pathogen Magnaporthe oryzae. Pias consists of a functionally specialized NLR pair, the helper Pias-1 and the sensor Pias-2, that is allelic to the previously characterized Pia pair of NLRs: the helper RGA4 and the sensor RGA5. Remarkably, Pias-2 carries a C-terminal DUF761 domain at a similar position to the heavy metal-associated (HMA) domain of RGA5. Phylogenomic analysis showed that Pias-2/RGA5 sensor NLRs have undergone recurrent genomic recombination within the genus Oryza, resulting in up to six sequence-divergent domain integrations. Allelic NLRs with divergent functions have been maintained transspecies in different Oryza lineages to detect sequence-divergent pathogen effectors. By contrast, Pias-1 has retained its NLR helper activity throughout evolution and is capable of functioning together with the divergent sensor-NLR RGA5 to respond to AVR-Pia. These results suggest that opposite selective forces have driven the evolution of paired NLRs: highly dynamic domain integration events maintained by balancing selection for sensor NLRs, in sharp contrast to purifying selection and functional conservation of immune signaling for helper NLRs.

Genome analyses reveal the hybrid origin of the staple crop white Guinea yam (Dioscorea rotundata).

White Guinea yam (Dioscorea rotundata) is an important staple tuber crop in West Africa. However, its origin remains unclear. In this study, we resequenced 336 accessions of white Guinea yam and compared them with the sequences of wild Dioscorea species using an improved reference genome sequence of D. rotundata In contrast to a previous study suggesting that D. rotundata originated from a subgroup of Dioscorea praehensilis, our results suggest a hybrid origin of white Guinea yam from crosses between the wild rainforest species D. praehensilis and the savannah-adapted species Dioscorea abyssinica We identified a greater genomic contribution from D. abyssinica in the sex chromosome of Guinea yam and extensive introgression around the SWEETIE gene. Our findings point to a complex domestication scenario for Guinea yam and highlight the importance of wild species as gene donors for improving this crop through molecular breeding.

RNA-Seq of in planta-expressed Magnaporthe oryzae genes identifies MoSVP as a highly expressed gene required for pathogenicity at the initial stage of infection.

The ascomycete fungus Magnaporthe oryzae is a hemibiotrophic pathogen that causes rice blast disease. Magnaporthe oryzae infects rice leaves, stems and panicles, and induces severe reductions in yield. Effector proteins secreted by M. oryzae in planta are thought to be involved its virulence activity. Here, using RNA-sequencing (RNA-Seq), we generated transcriptome data for M. oryzae isolate Ina168 during the initial stages of infection. We prepared samples from conidia (the inoculum) and from peeled epidermal cotyledon tissue of susceptible barley Hordeum vulgare 'Nigrate' at 12, 24, 36 and 48 hours post-inoculation (hpi). We also generated a draft genome sequence of M. oryzae isolate Ina168 and used it as a reference for mapping the RNA-Seq reads. Gene expression profiling across all stages of M. oryzae infection revealed 1728 putative secreted effector protein genes. We selected seven such genes that were strongly up-regulated at 12 hpi and down-regulated at 24 or 36 hpi and performed gene knockout analysis to determine their roles in pathogenicity. Knockout of MoSVP, encoding a small putative secreted protein with a hydrophobic surface binding protein A domain, resulted in a reduction in pathogenicity, suggesting that MoSVP is a novel virulence effector of M. oryzae.

MoST1 encoding a hexose transporter-like protein is involved in both conidiation and mycelial melanization of Magnaporthe oryzae.

In a large-scale gene disruption screen of Magnaporthe oryzae, a gene MoST1 encoding a protein belonging to the hexose transporter family was identified as a gene required for conidiation and culture pigmentation. The gene MoST1 located on chromosome V of the M. oryzae genome was predicted to be 1892 bp in length with two introns encoding a 547-amino-acid protein with 12 putative transmembrane domains. Targeted gene disruption of MoST1 resulted in a mutant (most1) with extremely poor conidiation and defects in colony melanization. These phenotypes were complemented by re-introduction of an intact copy of MoST1. We generated a transgenic line harboring a vector containing the MoST1 promoter fused with a reporter protein gene mCherry. The mCherry fluorescence was observed in mycelia, conidia, germ tubes, and appressoria in M. oryzae. There are 66 other hexose transporter-like genes in M. oryzae, and we performed complementation assay with three genes most closely related to MoST1. However, none of them complemented the most1 mutant in conidiation and melanization, indicating that the homologs do not complement the function of MoST1. These results suggest that MoST1 has a specific role for conidiation and mycelial melanization, which is not shared by other hexose transporter family of M. oryzae.

Deployment of the Burkholderia glumae type III secretion system as an efficient tool for translocating pathogen effectors to monocot cells.

Genome sequences of plant fungal pathogens have enabled the identification of effectors that cooperatively modulate the cellular environment for successful fungal growth and suppress host defense. Identification and characterization of novel effector proteins are crucial for understanding pathogen virulence and host-plant defense mechanisms. Previous reports indicate that the Pseudomonas syringae pv. tomato DC3000 type III secretion system (T3SS) can be used to study how non-bacterial effectors manipulate dicot plant cell function using the effector detector vector (pEDV) system. Here we report a pEDV-based effector delivery system in which the T3SS of Burkholderia glumae, an emerging rice pathogen, is used to translocate the AVR-Pik and AVR-Pii effectors of the fungal pathogen Magnaporthe oryzae to rice cytoplasm. The translocated AVR-Pik and AVR-Pii showed avirulence activity when tested in rice cultivars containing the cognate R genes. AVR-Pik reduced and delayed the hypersensitive response triggered by B. glumae in the non-host plant Nicotiana benthamiana, indicative of an immunosuppressive virulence activity. AVR proteins fused with fluorescent protein and nuclear localization signal were delivered by B. glumae T3SS and observed in the nuclei of infected cells in rice, wheat, barley and N. benthamiana. Our bacterial T3SS-enabled eukaryotic effector delivery and subcellular localization assays provide a useful method for identifying and studying effector functions in monocot plants.

Arms race co-evolution of Magnaporthe oryzae AVR-Pik and rice Pik genes driven by their physical interactions.

Attack and counter-attack impose strong reciprocal selection on pathogens and hosts, leading to development of arms race evolutionary dynamics. Here we show that Magnaporthe oryzae avirulence gene AVR-Pik and the cognate rice resistance (R) gene Pik are highly variable, with multiple alleles in which DNA replacements cause amino acid changes. There is tight recognition specificity of the AVR-Pik alleles by the various Pik alleles. We found that AVR-Pik physically binds the N-terminal coiled-coil domain of Pik in a yeast two-hybrid assay as well as in an in planta co-immunoprecipitation assay. This binding specificity correlates with the recognition specificity between AVR and R genes. We propose that AVR-Pik and Pik are locked into arms race co-evolution driven by their direct physical interactions.

Large-scale gene disruption in Magnaporthe oryzae identifies MC69, a secreted protein required for infection by monocot and dicot fungal pathogens.

To search for virulence effector genes of the rice blast fungus, Magnaporthe oryzae, we carried out a large-scale targeted disruption of genes for 78 putative secreted proteins that are expressed during the early stages of infection of M. oryzae. Disruption of the majority of genes did not affect growth, conidiation, or pathogenicity of M. oryzae. One exception was the gene MC69. The mc69 mutant showed a severe reduction in blast symptoms on rice and barley, indicating the importance of MC69 for pathogenicity of M. oryzae. The mc69 mutant did not exhibit changes in saprophytic growth and conidiation. Microscopic analysis of infection behavior in the mc69 mutant revealed that MC69 is dispensable for appressorium formation. However, mc69 mutant failed to develop invasive hyphae after appressorium formation in rice leaf sheath, indicating a critical role of MC69 in interaction with host plants. MC69 encodes a hypothetical 54 amino acids protein with a signal peptide. Live-cell imaging suggested that fluorescently labeled MC69 was not translocated into rice cytoplasm. Site-directed mutagenesis of two conserved cysteine residues (Cys36 and Cys46) in the mature MC69 impaired function of MC69 without affecting its secretion, suggesting the importance of the disulfide bond in MC69 pathogenicity function. Furthermore, deletion of the MC69 orthologous gene reduced pathogenicity of the cucumber anthracnose fungus Colletotrichum orbiculare on both cucumber and Nicotiana benthamiana leaves. We conclude that MC69 is a secreted pathogenicity protein commonly required for infection of two different plant pathogenic fungi, M. oryzae and C. orbiculare pathogenic on monocot and dicot plants, respectively.

Characterization of endo-1,3-1,4-ß-glucanases in GH family 12 from Magnaporthe oryzae.

We have cloned three putative endoglucanase cDNAs, designated MoCel12A, MoCel12B, and MoCel12C, from Magnaporthe oryzae. The deduced peptide sequences of both MoCel12A and MoCel12B contain secretion signal peptides and a catalytic core domain that classify them into GH subfamily 12-1. In contrast, the deduced peptide sequence of MoCel12C consists of a signal peptide, a catalytic core domain, and a fungal-type carbohydrate binding module belonging to GH subfamily 12-2. Although most GH family 12 endoglucanases hydrolyze ß-1,4-glucans such as carboxymethylcellulose or phosphoric acid-swollen cellulose, MoCel12A that was prepared by overexpression in M. oryzae and Brevibacillus choshinensis hydrolyzed specifically 1,3-1,4-ß-glucans, such as barley ß-glucan and lichenan. The specific activity of MoCel12A overexpressed in M. oryzae was about 20 times higher than that prepared from B. choshinensis. Furthermore, MoCel12B prepared by overexpression in B. choshinensis also revealed preferential hydrolysis of endo-1,3-1,4-ß-glucans with limited hydrolysis on carboxymethylcellulose. In comparison with MoCel12A, the activity of MoCel12B was more stable under alkaline conditions. Levels of mRNA encoding MoCel12A were constitutively high during infection and spore formation. The overexpression and disruption of the MoCel12A gene did not affect germination, appressorium formation, or invasion rate; however, M. oryzae overexpressing MoCel12A produced larger numbers of spores than the wild type or a mutant in which the MoCel12A gene was disrupted. These results suggest that MoCel12A functions in part to hydrolyze 1,3-1,4-ß-glucan during infection and spore formation.