CRISPR/SpCas9-mediated double knockout of barley Microrchidia MORC1 and MORC6a reveals their strong involvement in plant immunity,transcriptional gene silencing and plant growth

The Microrchidia (MORC) family proteins are important nuclear regulators in both animals and plants with critical roles in epigenetic gene silencing and genome stabilization. In the crop plant barley (Hordeum vulgare), seven MORC gene family members have been described. While barley HvMORC1 has been functionally characterized, very little information is available about other HvMORC paralogs. In this study, we elucidate the role of HvMORC6a and its potential interactors in regulating plant immunity via analysis of CRISPR/SpCas9-mediated single and double knockout (dKO) mutants, hvmorc1 (previously generated and characterized by our group), hvmorc6a, and hvmorc1/6a. For generation of hvmorc1/6a, we utilized two different strategies: (i) successive Agrobacterium-mediated transformation of homozygous single mutants, hvmorc1 and hvmorc6a, with the respective second construct, and (ii) simultaneous transformation with both hvmorc1 and hvmorc6a CRISPR/SpCas9 constructs. Total mutation efficiency in transformed homozygous single mutants ranged from 80 to 90%, while upon simultaneous transformation, SpCas9-induced mutation in both HvMORC1 and HvMORC6a genes was observed in 58% of T0 plants. Subsequent infection assays showed that HvMORC6a covers a key role in resistance to biotrophic (Blumeria graminis) and necrotrophic (Fusarium graminearum) plant pathogenic fungi, where the dKO hvmorc1/6a showed the strongest resistant phenotype. Consistent with this, the dKO showed highest levels of basal PR gene expression and derepression of TEs. Finally, we demonstrate that HvMORC1 and HvMORC6a form distinct nucleocytoplasmic homo-/heteromers with other HvMORCs and interact with components of the RNA-directed DNA methylation (RdDM) pathway, further substantiating that MORC proteins are involved in the regulation of TEs in barley.     

Inducible expression of a fusion gene encoding two proteinase inhibitors leads to insect and pathogen resistance in transgenic rice

Plant proteinase inhibitors (PIs) are considered as candidates for increased insect resistance in transgenic plants. Insect adaptation to PI ingestion might, however, compromise the benefits received by transgenic expression of PIs. In this study, the maize proteinase inhibitor (MPI), an inhibitor of insect serine proteinases, and the potato carboxypeptidase inhibitor (PCI) were fused into a single open reading frame and introduced into rice plants. The two PIs were linked using either the processing site of the Bacillus thuringiensis Cry1B precursor protein or the 2A sequence from the foot‐and‐mouth disease virus (FMDV). Expression of each fusion gene was driven by the wound‐ and pathogen‐inducible mpi promoter. The mpi‐pci fusion gene was stably inherited for at least three generations with no penalty on plant phenotype. An important reduction in larval weight of Chilo suppressalis fed on mpi‐pci rice, compared with larvae fed on wild‐type plants, was observed. Expression of the mpi‐pci fusion gene confers resistance to C. suppressalis (striped stem borer), one of the most important insect pest of rice. The mpi‐pci expression systems described may represent a suitable strategy for insect pest control, better than strategies based on the use of single PI genes, by preventing insect adaptive responses. The rice plants expressing the mpi‐pci fusion gene also showed enhanced resistance to infection by the fungus Magnaporthe oryzae, the causal agent of the rice blast disease. Our results illustrate the usefulness of the inducible expression of the mpi‐pci fusion gene for dual resistance against insects and pathogens in rice plants.     

Effects of temperature and medium composition on inhibitory activities of gossypol‐related compounds against aflatoxigenic fungi

Aims

To investigate the effects of temperature and medium composition on growth/aflatoxin inhibitory activities of terpenoids gossypol, gossypolone and apogossypolone against Aspergillus flavus and A. parasiticus.

Methods and Results

The compounds were tested at a concentration of 100 μg ml^?1 in a Czapek Dox (Czapek) agar medium at 25, 31 and 37°C. Increased incubation temperature marginally increased growth inhibition caused by these compounds, but reduced the aflatoxin inhibition effected by gossypol. Gossypolone and apogossypolone retained good aflatoxin inhibitory activity against A. flavus and A. parasiticus at higher incubation temperatures. However, increased temperature also significantly reduced aflatoxin production in control cultures. The effects of the terpenoids on fungal growth and aflatoxin production against the same fungi were also determined in Czapek, Czapek with a protein/amino acid addendum and yeast extract sucrose (YES) media. Growth of these fungi in the protein‐supplemented Czapek medium or in the YES medium greatly reduced the growth inhibition effects of the terpenoids. Apogossypolone displayed strong anti‐aflatoxigenic activity in the Czapek medium, but this activity was significantly reduced in the protein‐amended Czapek and YES media. Gossypol, which displayed little to no aflatoxin inhibitory activity in the Czapek medium, did yield significant anti‐aflatoxigenic activity in the YES medium.

Conclusions

Incubation temperature and media composition are important parameters involved in the regulation of aflatoxin production in A. flavus and A. parasiticus. These parameters also affect the potency of growth and aflatoxin inhibitory activities of these gossypol‐related compounds against aflatoxigenic fungi.

Significance and Impact of the Study

Studies utilizing gossypol‐related compounds as inhibitory agents of biological activities should be interpreted with caution due to compound interaction with multiple components of the test system, especially serum proteins.     

Imbalance of tyrosine by modulating TyrA arogenate dehydrogenases impacts growth and development of Arabidopsis thaliana

l ‐Tyrosine is an essential aromatic amino acid required for the synthesis of proteins and a diverse array of plant natural products; however, little is known on how the levels of tyrosine are controlled in planta and linked to overall growth and development. Most plants synthesize tyrosine by TyrA arogenate dehydrogenases, which are strongly feedback‐inhibited by tyrosine and encoded by TyrA1 and TyrA2 genes in Arabidopsis thaliana. While TyrA enzymes have been extensively characterized at biochemical levels, their in planta functions remain uncertain. Here we found that TyrA1 suppression reduces seed yield due to impaired anther dehiscence, whereas TyrA2 knockout leads to slow growth with reticulate leaves. The tyra2 mutant phenotypes were exacerbated by TyrA1 suppression and rescued by the expression of TyrA2, TyrA1 or tyrosine feeding. Low‐light conditions synchronized the tyra2 and wild‐type growth, and ameliorated the tyra2 leaf reticulation. After shifting to normal light, tyra2 transiently decreased tyrosine and subsequently increased aspartate before the appearance of the leaf phenotypes. Overexpression of the deregulated TyrA enzymes led to hyper‐accumulation of tyrosine, which was also accompanied by elevated aspartate and reticulate leaves. These results revealed that TyrA1 and TyrA2 have distinct and overlapping functions in flower and leaf development, respectively, and that imbalance of tyrosine, caused by altered TyrA activity and regulation, impacts growth and development of Arabidopsis. The findings provide critical bases for improving the production of tyrosine and its derived natural products, and further elucidating the coordinated metabolic and physiological processes to maintain tyrosine levels in plants.     

Yucasin is a potent inhibitor of YUCCA,a key enzyme in auxin biosynthesis

Indole‐3–acetic acid (IAA), an auxin plant hormone, is biosynthesized from tryptophan. The indole‐3–pyruvic acid (IPyA) pathway, involving the tryptophan aminotransferase TAA1 and YUCCA (YUC) enzymes, was recently found to be a major IAA biosynthetic pathway in Arabidopsis. TAA1 catalyzes the conversion of tryptophan to IPyA, and YUC produces IAA from IPyA. Using a chemical biology approach with maize coleoptiles, we identified 5–(4–chlorophenyl)‐4H‐1,2,4–triazole‐3–thiol (yucasin) as a potent inhibitor of IAA biosynthesis in YUC‐expressing coleoptile tips. Enzymatic analysis of recombinant AtYUC1‐His suggested that yucasin strongly inhibited YUC1‐His activity against the substrate IPyA in a competitive manner. Phenotypic analysis of Arabidopsis YUC1 over‐expression lines (35S::YUC1) demonstrated that yucasin acts in IAA biosynthesis catalyzed by YUC. In addition, 35S::YUC1 seedlings showed resistance to yucasin in terms of root growth. A loss‐of‐function mutant of TAA1, sav3–2, was hypersensitive to yucasin in terms of root growth and hypocotyl elongation of etiolated seedlings. Yucasin combined with the TAA1 inhibitor l –kynurenine acted additively in Arabidopsis seedlings, producing a phenotype similar to yucasin‐treated sav3–2 seedlings, indicating the importance of IAA biosynthesis via the IPyA pathway in root growth and leaf vascular development. The present study showed that yucasin is a potent inhibitor of YUC enzymes that offers an effective tool for analyzing the contribution of IAA biosynthesis via the IPyA pathway to plant development and physiological processes.     

金属离子和氨基酸对Eupenicillium sp.E-UN41生物合成咪唑立宾的影响

为考察金属离子和氨基酸对Eupenicillium sp.E-UN41菌体生长及其产生的次级代谢产物咪唑立宾(MZ)的生物合成影响,在培养基中添加了无机盐和氨基酸.结果表明在发酵培养基中添加适量的镁、钠、锰、钾、钙等金属离子可提高咪唑立宾产量5％～15％,添加1.0％L-组氨酸,MZ产量提高94％.添加适宜的L-天门冬氨酸和L-甘氨酸对产MZ亦有促进作用,而L-精氨酸明显抑制Eupenicillium sp.E-UN41生物合成MZ.本研究为咪唑立宾发酵的产业化奠定了基础.     

Effects of rare earth elements on growth rate,lipids, fatty acids and pigments in microalgae

The extensive exploitation of rare earth elements (REEs), particularly in electronic technologies and agriculture has concomitantly raised the environmental load. Their resulting effects on primary producers such as microalgae are, however, poorly understood. We have studied these effects on two microalgae of biotechnological interest. The yellow‐green alga Trachydiscus minutus (Eustigmatophyceae, Ochrophyta), and the green alga Parachlorella kessleri (Trebouxiophyceae, Chlorophyta) were cultivated in mineral medium supplemented with 10 μmol L^?1 chlorides of REEs: cerium, gadolinium, lanthanum, lutetium, praseodymium, scandium, and with monazite, which is a mineral rich in those elements. We observed growth rates at different mean light intensities (20, 50, 150 and 300 μmol m^?2 s^?1). The high growth rate of P. kessleri was not affected by the presence of any lanthanide, and decreased proportionally with light intensity (from 0.2 to 0.04 doublings per hour). In contrast, the growth rate of T. minutus was about three times lower compared with P. kessleri, with an optimum at 50 μmol m^?2 s^?1 and decreased at higher or lower light intensities. In the presence of Ce³⁺, La³⁺ and Sc³⁺, the growth rate markedly increased to a value that corresponded to the growth rate in P. kessleri at the same light intensity. The composition and content of pigments and lipids were followed at the optimum light intensity for both species. The lipid content (percentage of dry weight) varied only slightly in the presence of individual rare earths. There was, however, an increase in saturated fatty acids at the expense of polyunsaturated fatty acids. The effect of REEs on pigments was variable: the presence of Ce³⁺, Gd³⁺, La³⁺ and Sc³⁺ caused an increase in the concentrations of major pigments such as lutein, violaxanthin, β‐carotene or chlorophylls, while Pr³⁺ and Lu³⁺ reduced them.     

Negative and positive interactions among plants: effects of competitors and litter on seedling emergence and growth of forest and grassland species

Living plant neighbours, but also their dead aboveground remains (i.e. litter), may individually exert negative or positive effects on plant recruitment. Although living plants and litter co‐occur in most ecosystems, few studies have addressed their combined effects, and conclusions are ambivalent. Therefore, we examined the response in terms of seedling emergence and growth of herbaceous grassland and forest species to different litter types and amounts and the presence of competitors. We conducted a pot experiment testing the effects of litter type (grass, oak), litter amount (low, medium, high) and interspecific competition (presence or absence of four Festuca arundinacea individuals) on seedling emergence and biomass of four congeneric pairs of hemicryptophytes from two habitat types (woodland, grassland). Interactions between litter and competition were weak. Litter presence increased competitor biomass. It also had positive effects on seedling emergence at low litter amounts and negative effects at high litter amounts, while competition had no effect on seedling emergence. Seedling biomass was negatively affected by the presence of competitors, and this effect was stronger in combination with high amounts of litter. Litter affected seedling emergence while competition determined the biomass of the emerged individuals, both affecting early stages of seedling recruitment. High litter accumulation also reduced seedling biomass, but this effect seemed to be additive to competitor effects. This suggests that live and dead plant mass can affect species recruitment in natural systems, but the mechanisms by which they operate and their timing differ.     

Effects of cyanobacteria,fish kairomones,and the presence of ostracods on the demography of Simocephalus vetulus (Cladocera)

Simocephalus vetulus is a large (2.0–4.0 mm at maturity) cladoceran often found in the littoral region of lakes and ponds, and capable of moderate growth rates even on poor‐quality cyanobacterial diets. It frequently co‐occurs with fishes and similar sized ostracods such as Heterocypris incongruens, but little is known of its response to fish kairomones or its interactions with potential competitors. We studied the demographic responses of S. vetulus fed the green alga Scenedesmus acutus, Microcystis cf. aeruginosa strain A, Microcystis cf. aeruginosa strain B, or Limnothrix sp. Experiments were conducted separately and together in the presence of Heterocypris incongruens and cichlid fish (Oreochromis) kairomones. A diet of Limnothrix sp. resulted in the lowest population growth rate (0.21±0.023 d^?1), while on diets of S. acutus or Microcystis, population growth was higher (0.30±0.009 d^?1). The presence of ostracods resulted in significantly higher growth rates of S. vetulus fed Limnothrix (0.33±0.01 d^?1), but not Microcystis or S. acutus. Regardless of the diet, the presence of fish kairomones resulted in significantly higher growth rates as compared with controls, particularly when ostracods were also present. Coexistence with ostracods may be beneficial to S. vetulus, particularly when food quality is poor.     

Effects of salt stress and rhizobial inoculation on growth and nitrogen fixation of three peanut cultivars

Increasing soil salinity represents a major constraint for agriculture in arid and semi‐arid lands, where mineral nitrogen (N) deficiency is also a frequent characteristic of soils. Biological N fixation by legumes may constitute a sustainable alternative to chemical fertilisation in salinity‐affected areas, provided that adapted cultivars and inoculants are available. Here, the performance of three peanut cultivars nodulated with two different rhizobial strains that differ in their salt tolerance was evaluated under moderately saline water irrigation and compared with that of N‐fertilised plants. Shoot weight was used as an indicator of yield. Under non‐saline conditions, higher yields were obtained using N fertilisation rather than inoculation for all the varieties tested. However, under salt stress, the yield of inoculated plants became comparable to that of N‐fertilised plants, with minor differences depending on the peanut cultivar and rhizobial strain. Our results indicate that N fixation might represent an economical, competitive and environmentally friendly choice with respect to mineral N fertilisation for peanut cultivation under moderate saline conditions.     

Overexpression of Populus trichocarpa CYP85A3 promotes growth and biomass production in transgenic trees

Brassinosteroids (BRs) are essential hormones that play crucial roles in plant growth, reproduction and response to abiotic and biotic stress. In Arabidopsis, AtCYP85A2 works as a bifunctional cytochrome P450 monooxygenase to catalyse the conversion of castasterone to brassinolide, a final rate‐limiting step in the BR‐biosynthetic pathway. Here, we report the functional characterizations of PtCYP85A3, one of the three AtCYP85A2 homologous genes from Populus trichocarpa. PtCYP85A3 shares the highest similarity with AtCYP85A2 and can rescue the retarded‐growth phenotype of the Arabidopsis cyp85a2‐2 and tomato d^x mutants. Constitutive expression of PtCYP85A3, driven by the cauliflower mosaic virus 35S promoter, increased the endogenous BR levels and significantly promoted the growth and biomass production in both transgenic tomato and poplar. Compared to the wild type, plant height, shoot fresh weight and fruit yield increased 50%, 56% and 43%, respectively, in transgenic tomato plants. Similarly, plant height and stem diameter increased 15% and 25%, respectively, in transgenic poplar plants. Further study revealed that overexpression of PtCYP85A3 enhanced xylem formation without affecting the composition of cellulose and lignin, as well as the cell wall thickness in transgenic poplar. Our finding suggests that PtCYP85A3 could be used as a potential candidate gene for engineering fast‐growing trees with improved wood production.     

Effects of Rag1 aphid‐resistant soybean on mortality,development, and preference of brown marmorated stink bug

The brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), poses a new threat to soybean, Glycine max (L.) Merrill (Fabaceae), production in the north central USA. As H. halys continues to spread and increase in abundance in the region, the interaction between H. halys and management tactics deployed for other pests must be determined. Currently, the soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is the most abundant and damaging insect pest of soybean in the region. Aphid‐resistant soybean, mainly with the Rag1 gene, is commercially available for management of A. glycines. Here, experiments were performed to evaluate the effects of Rag1 aphid‐resistant soybean on the mortality, development, and preference of H. halys. In a no‐choice test, mortality of H. halys reared on Rag1 aphid‐resistant soybean pods was significantly lower than when reared on aphid‐susceptible soybean pods (28 vs. 53%). Development time, adult weight, and proportion females of surviving adults did not differ when reared on Rag1 aphid‐resistant or aphid‐susceptible soybean pods. In choice tests, H. halys exhibited a preference for Rag1 aphid‐resistant over aphid‐susceptible soybean pods after 4 h, but not after 24 h. Halyomorpha halys exhibited no preference when tested with vegetative‐stage or reproductive‐stage soybean plants. The preference by H. halys for Rag1 aphid‐resistant soybean pods and the decreased mortality when reared on these pods suggests that the use of Rag1 aphid‐resistant soybean may favor this emerging pest in the north central USA.