April 21, 2020  |  

Cichorium intybus L.?×?Cicerbita alpina Walbr.: doubled haploid chicory induction and CENH3 characterization

Intergeneric hybridization between industrial chicory (Cichorium intybus L.) and Cicerbita alpina Walbr. induces interspecific hybrids and haploid chicory plants after in vitro embryo rescue. The protocol yielded haploids in 5 out of 12 cultivars pollinated; altogether 18 haploids were regenerated from 2836 embryos, with a maximum efficiency of 1.96% haploids per cross. Obtained haploids were chromosome doubled with mitosis inhibitors trifluralin and oryzalin; exposure to 0.05 g L-1 oryzalin during one week was the most efficient treatment to regenerate doubled haploids. Inbreeding effects in vitro were limited, but the ploidy level affects morphology. Transcriptome sequencing revealed two unique copies of CENH3 in Cicerbita alpina Walbr. Comparison of CENH3.1 protein sequences of Cicerbita and Cichorium obtained through transcriptome and whole shotgun genome sequencing revealed two amino-acid substitutions at critical residues of the histone fold domain. These particular changes cause chromosome elimination and reduced centromere loading in several other species and might indicate a CENH3-dependent mechanism causing chromosome elimination of parental chromosomes during Cichorium?×?Cicerbita intergeneric hybridization. Our results provide insights in chromosome elimination and might increase the efficiency of haploid induction in Cichorium.

April 21, 2020  |  

Adaptive Strategies in a Poly-Extreme Environment: Differentiation of Vegetative Cells in Serratia ureilytica and Resistance to Extreme Conditions.

Poly-extreme terrestrial habitats are often used as analogs to extra-terrestrial environments. Understanding the adaptive strategies allowing bacteria to thrive and survive under these conditions could help in our quest for extra-terrestrial planets suitable for life and understanding how life evolved in the harsh early earth conditions. A prime example of such a survival strategy is the modification of vegetative cells into resistant resting structures. These differentiated cells are often observed in response to harsh environmental conditions. The environmental strain (strain Lr5/4) belonging to Serratia ureilytica was isolated from a geothermal spring in Lirima, Atacama Desert, Chile. The Atacama Desert is the driest habitat on Earth and furthermore, due to its high altitude, it is exposed to an increased amount of UV radiation. The geothermal spring from which the strain was isolated is oligotrophic and the temperature of 54°C exceeds mesophilic conditions (15 to 45°C). Although the vegetative cells were tolerant to various environmental insults (desiccation, extreme pH, glycerol), a modified cell type was formed in response to nutrient deprivation, UV radiation and thermal shock. Scanning (SEM) and Transmission Electron Microscopy (TEM) analyses of vegetative cells and the modified cell structures were performed. In SEM, a change toward a circular shape with reduced size was observed. These circular cells possessed what appears as extra coating layers under TEM. The resistance of the modified cells was also investigated, they were resistant to wet heat, UV radiation and desiccation, while vegetative cells did not withstand any of those conditions. A phylogenomic analysis was undertaken to investigate the presence of known genes involved in dormancy in other bacterial clades. Genes related to spore-formation in Myxococcus and Firmicutes were found in S. ureilytica Lr5/4 genome; however, these genes were not enough for a full sporulation pathway that resembles either group. Although, the molecular pathway of cell differentiation in S. ureilytica Lr5/4 is not fully defined, the identified genes may contribute to the modified phenotype in the Serratia genus. Here, we show that a modified cell structure can occur as a response to extremity in a species that was previously not known to deploy this strategy. This strategy may be widely spread in bacteria, but only expressed under poly-extreme environmental conditions.

April 21, 2020  |  

Linking CRISPR-Cas9 interference in cassava to the evolution of editing-resistant geminiviruses.

Geminiviruses cause damaging diseases in several important crop species. However, limited progress has been made in developing crop varieties resistant to these highly diverse DNA viruses. Recently, the bacterial CRISPR/Cas9 system has been transferred to plants to target and confer immunity to geminiviruses. In this study, we use CRISPR-Cas9 interference in the staple food crop cassava with the aim of engineering resistance to African cassava mosaic virus, a member of a widespread and important family (Geminiviridae) of plant-pathogenic DNA viruses.Our results show that the CRISPR system fails to confer effective resistance to the virus during glasshouse inoculations. Further, we find that between 33 and 48% of edited virus genomes evolve a conserved single-nucleotide mutation that confers resistance to CRISPR-Cas9 cleavage. We also find that in the model plant Nicotiana benthamiana the replication of the novel, mutant virus is dependent on the presence of the wild-type virus.Our study highlights the risks associated with CRISPR-Cas9 virus immunity in eukaryotes given that the mutagenic nature of the system generates viral escapes in a short time period. Our in-depth analysis of virus populations also represents a template for future studies analyzing virus escape from anti-viral CRISPR transgenics. This is especially important for informing regulation of such actively mutagenic applications of CRISPR-Cas9 technology in agriculture.

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