Induction of defense mechanisms in seedlings of oilseed winter rape inoculated with Phoma lingam (Leptosphaeria maculans)

The aim of this study was to find the biochemical markers of blackleg, evoked by the fungus Leptosphaeria maculans in seedlings of oilseed winter rape. Analyses of antioxidant enzymes activity and reactive oxygen species detection were performed 24, 48 and 72 h after inoculation. Moreover, measurements of the total pool of phenolic compounds, trans-cinnamic acid and ferulic acid were carried out. The results proved the virulence of Ph L5 isolate towards the rape seedlings. An increase in total phenolic content, including trans-cinnamic acid, was observed 24 h after inoculation. High activity of phenylalanine ammonia-lyase is probably another symptom of the defense mechanisms, despite the fact that it was not accompanied by elevated phenolics level, which were probably rapidly exhausted during Phoma lingam inoculation. A drop in phenolics content was balanced by higher activity of SOD, CAT and non-specific peroxidases. Enhanced activity of these enzymes was a response to the reactive oxygen species overproduction. ROS accumulation in indirectly inoculated first true leaves after inoculation may suggest a systemic defense response.     

A simple and efficient CRISPR/Cas9 platform for induction of single and multiple,heritable mutations in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

Background

Genome editing of monocot plants can be accomplished by using the components of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR associated Cas9) technology specifically optimized for these types of plants. Here, we present the development of RNA-guided Cas9 system for simplex and multiplex genome editing in barley.

Results

We developed a set of customizable RNA-guided Cas9 binary vectors and sgRNA modules for simplex and multiplex editing in barley. To facilitate the design of RNA-guided Cas9 constructs, the pBract derived binary vectors were adapted to Gateway cloning and only one restriction enzyme was required for construction of the sgRNA. We designed a synthetic, codon optimized Cas9 gene containing the N terminal SV40 nuclear localization signal and the UBQ10 Arabidopsis 1st intron. Two different sgRNAs were constructed for simplex editing and one polycistronic tRNA-gRNA construct (PTG) for multiplex editing using an endogenous tRNA processing system. The RNA-guided Cas9 constructs were validated in transgenic barley plants produced by Agrobacterium-mediated transformation. The highest mutation rate was observed in simplex editing of the cytokinin oxidase/dehydrogenase HvCKX1 gene, where mutations at the hvckx1 locus were detected in 88% of the screened T₀ plants. We also proved the efficacy of the PTG construct in the multiplex editing of two CKX genes by obtaining 9 plants (21% of all edited plants) with mutations induced in both HvCKX1 and HvCKX3. Analysis of the T₁ lines revealed that mutations in the HvCKX1 gene were transmitted to the next generation of plants. Among 220 screened T₁ plants we identified 85 heterozygous and 28 homozygous mutants, most of them bearing frameshift mutations in the HvCKX1 gene. We also observed independent segregation of mutations and the Cas9-sgRNA T-DNA insert in several T₁ plants. Moreover, the knockout mutations of the Nud gene generated phenotype mutants with naked grains, and the phenotypic changes were identifiable in T₀ plants.

Conclusions

We demonstrated the effectiveness of an optimized RNA-guided Cas9 system that can be used for generating homozygous knockout mutants in the progeny of transgenic barely plants. This is also the first report of successful multiplex editing in barley using a tRNA processing system.

     

Circadian dynamics of locomotor activity and deltamethrin susceptibility in the pine weevil,Hylobius abietis

Rhythmic locomotor activity and daily susceptibility to deltamethrin were tested in the pine weevil,Hylobius abietis (L.) (Coleoptera, Curculionidae), a pest of young conifer plants. In constant darkness, beetles revealed a free-running circadian pattern of locomotor activity (average period 22h : 20min). Under long photoperiod, L:D 18:6, entrainment of motor activity was observed. In the entrained population of the weevils, a peak of locomotor activity occurred at about the beginning of the dark phase, and the minimum occurred in the middle of the light phase. Fluctuations of susceptibility to a standard dose of deltamethrin (0.5 μg/g body weight) varied across the day, and were inversely correlated to the changes in locomotor activity. The importance of considering the daily organization of insect biology in studying insect resistance to pesticides is re-emphasized. 3 www.csa.com. The following databases were screened: Animal Behaviour Abstracts, AGRICOLA, Aquatic Science and Fishery Abstracts, Biological Sciences, Ecology Abstracts, Entomology Abstracts, Environmental Science and Pollution Abstracts, Health and Safety Science Abstracts, Pollution Abstracts, Water Resource Abstracts, Toxicology Abstracts, TOXLINE.