Knockout of MITOGEN-ACTIVATED PROTEIN KINASE 3 causes barley root resistance against Fusarium graminearum.

The roles of mitogen-activated protein kinases (MAPKs) in plant-fungal pathogenic interactions are poorly understood in crops. Here, microscopic, phenotypic, proteomic, and biochemical analyses revealed that roots of independent transcription activator-like effector nuclease (TALEN)-based knockout lines of barley (Hordeum vulgare L.) MAPK 3 (HvMPK3 KO) were resistant against Fusarium graminearum infection. When co-cultured with roots of the HvMPK3 KO lines, F. graminearum hyphae were excluded to the extracellular space, the growth pattern of extracellular hyphae was considerably deregulated, mycelia development was less efficient, and number of appressoria-like structures and their penetration potential were substantially reduced. Intracellular penetration of hyphae was preceded by the massive production of reactive oxygen species (ROS) in attacked cells of the wild-type (WT), but ROS production was mitigated in the HvMPK3 KO lines. Suppression of ROS production in these lines coincided with elevated abundance of catalase (CAT) and ascorbate peroxidase (APX). Moreover, differential proteomic analysis revealed downregulation of several defense-related proteins in WT, and the upregulation of pathogenesis-related protein 1 (PR-1) and cysteine proteases in HvMPK3 KO lines. Proteins involved in suberin formation, such as peroxidases, lipid transfer proteins (LTPs), and the GDSL esterase/lipase (containing "GDSL" aminosequence motif) were differentially regulated in HvMPK3 KO lines after F. graminearum inoculation. Consistent with proteomic analysis, microscopic observations showed enhanced suberin accumulation in roots of HvMPK3 KO lines, most likely contributing to the arrested infection by F. graminearum. These results suggest that TALEN-based knockout of HvMPK3 leads to barley root resistance against Fusarium root rot.

Post-Embryonic Lateral Organ Development and Adaxial-Abaxial Polarity Are Regulated by the Combined Effect of ENHANCER OF SHOOT REGENERATION 1 and WUSCHEL in Arabidopsis Shoots.

The development of above-ground lateral organs is initiated at the peripheral zone of the shoot apical meristem (SAM). The coordination of cell fate determination and the maintenance of stem cells are achieved through a complex regulatory network comprised of transcription factors. Two AP2/ERF transcription factor family genes, ESR1/DRN and ESR2/DRNL/SOB/BOL, regulate cotyledon and flower formation and de novo organogenesis in tissue culture. However, their roles in post-embryonic lateral organ development remain elusive. In this study, we analyzed the genetic interactions among SAM-related genes, WUS and STM, two ESR genes, and one of the HD-ZIP III members, REV, whose protein product interacts with ESR1 in planta. We found that esr1 mutations substantially enhanced the wus and stm phenotypes, which bear a striking resemblance to those of the wus rev and stm rev double mutants, respectively. Aberrant adaxial-abaxial polarity is observed in wus esr1 at relatively low penetrance. On the contrary, the esr2 mutation partially suppressed stm phenotypes in the later vegetative phase. Such complex genetic interactions appear to be attributed to the distinct expression pattern of two ESR genes because the ESR1 promoter-driving ESR2 is capable of rescuing phenotypes caused by the esr1 mutation. Our results pose the unique genetic relevance of ESR1 and the SAM-related gene interactions in the development of rosette leaves.

Interpreting Cytokinin Action as Anterograde Signaling and Beyond.

Among the major phytohormones, the cytokinin exhibits unique features for its ability to positively affect the developmental status of plastids. Even early on in its research, cytokinins were known to promote plastid differentiation and to reduce the loss of chlorophyll in detached leaves. Since the discovery of the components of cytokinin perception and primary signaling, the genes involved in photosynthesis and plastid differentiation have been identified as those directly targeted by type-B response regulators. Furthermore, cytokinins are known to modulate versatile cellular processes such as promoting the division and differentiation of cells and, in concert with auxin, initiating the de novo formation of shoot apical meristem (SAM) in tissue cultures. Yet how cytokinins precisely participate in such diverse cellular phenomena, and how the associated cellular processes are coordinated as a whole, remains unclear. A plausible presumption that would account for the coordinated gene expression is the tight and reciprocal communication between the nucleus and plastid. The fact that cytokinins affect plastid developmental status via gene expression in both the nucleus and plastid is interpreted here to suggest that cytokinin functions as an initiator of anterograde (nucleus-to-plastid) signaling. Based on this viewpoint, we first summarize the physiological relevance of cytokinins to the coordination of plastid differentiation with de novo shoot organogenesis in tissue culture systems. Next, the role of endogenous cytokinins in influencing plastid differentiation within the SAM of intact plants is discussed. Finally, a presumed plastid-derived signal in response to cytokinins for coupled nuclear gene expression is proposed.

CRISPR/Cas9 genome editing in ergot fungus Claviceps purpurea.

Claviceps purpurea is a filamentous fungus well known as a widespread plant pathogen, but it is also an important ergot alkaloid producer exploited by the pharmaceutic industry. In this work, we demonstrated that CRISPR/Cas9 can be a tool for directed mutagenesis in C. purpurea targeting pyr4 and TrpE genes encoding the orotidine 5'-phosphate decarboxylase involved in pyrimidine biosynthesis and the α-subunit of the anthranilate synthase involved in tryptophan biosynthesis, respectively. After protoplast transformation and single spore isolation, homokaryotic mutants showing uridine or tryptophan auxotrophy were selected. In all cases, insertions or insertions combined with deletions were found mostly 3 bp upstream of the PAM sequence. However, transformation efficiencies of CRISPR/Cas9 and CRISPR/Cas9 mediated homology-directed repair only slightly improved in comparison to homologous recombination-mediated knocking-out of the TrpE gene. Moreover, Trp auxotrophs were non-infectious towards rye plants likely due to a decreased production of the plant hormones auxins, which are synthesized by C. purpurea from indole-3-glycerolphosphate in Trp-dependent and Trp-independent biosynthetic pathways, and help the fungus to colonize the plant host. It was demonstrated that the CRISPR/Cas9 vector containing autonomous replicative sequence AMA1 can be fully removed by further culturing of C. purpurea on non-selective media. This method enables introducing multiple mutations in Claviceps and makes feasible metabolic engineering of industrial strains.

Overexpression of Trp-related genes in Claviceps purpurea leading to increased ergot alkaloid production.

The parasitic fungus Claviceps purpurea has been used for decades by the pharmaceutical industry as a valuable producer of ergot alkaloids. As the biosynthetic pathway of ergot alkaloids involves a common precursor L-tryptophan, targeted genetic modification of the related genes may improve production yield. In this work, the S76L mutated version of the trpE gene encoding anthranilate synthase was constitutively overexpressed in the fungus with the aim of overcoming feedback inhibition of the native enzyme by an excess of tryptophan. In another approach, the dmaW gene encoding dimethylallyltryptophan synthase, which produces a key intermediate for the biosynthesis of ergot alkaloids, was also constitutively overexpressed. Each of the above manipulations led to a significant increase (up to 7-fold) in the production of ergot alkaloids in submerged cultures.

[Monitoring of formation of diclofenac sodium salt hydrates and their influence on the drug dissolution from prepared tablets]. / Sledování vzniku hydrátu diklofenaku sodné soli a jejich vlivu na disoluci léciva z pripravených tablet.

NIR spectroscopy together with multivariate data analysis were used to analyze the hydrates of diclofenac sodium prepared from the non-aqueous solvents tetrahydrofuran and methanol under standard laboratory conditions at 20 °C and relative humidity less than 60%. It was confirmed that the developed PLS regression model can monitor the process of formation of hydrates. It was also found that the hydrated form of diclofenac sodium arises during the preparation of the dosage form the using technology of impregnating the solid carrier by non-aqueous solvents, which resulted in reducing of the drug release rate from prepared tablets up to twice. NIR spectroscopy was confirmed as one of the effective PAT (Process Analytical Technology) methods.