Z.F. Wang

Identification of Circular RNAs in Kiwifruit and Their Species-Specific Response to Bacterial Canker Pathogen Invasion

Research studies have recently focused on circle RNAs (circRNAs) in relation to their regulatory functions in animals. However, the systematic identification of circRNAs in plants, especially non-model plants, is limited. In addition, raw report on the prediction of the potential role of circRNAs in plant response to pathogen invasion is currently available. We conducted the systematic identification of circRNAs from four materials originating from three species belonging to genus Actinidia under different situations using ribosomal RNA (rRNA) depleted RNA-Seq data. A total of 3,582 circRNAs were identified in Actinidia, of which 64.01, 21.44, and 14.55% were intergenic circRNAs, exonic circRNAs, and intronic circRNAs, respectively. Tissue-specific expression of circRNAs was observed in kiwifruit, and a species-specific response was detected when infected with Pseudomonas syringae pv. actinidiae (Psa), which is the causative agent of kiwifruit bacterial canker disease. Furthermore, we found that both exonic and intronic circRNAs were significantly positively correlated to parent protein-coding genes, and intronic circRNAs are a class of highly remarkable regulators the parent genes comparing to that of exonic circRNAs. Expression and weighted gene co-expression network analysis (WGCNA) identified a set of circRNAs that were closely associated with plant defense response. The findings of the presents study suggest that circRNAs exhibit tissue- and species-specific expression, as well as play an important role in plant immune response.

Optimized paired-sgRNA/Cas9 cloning and expression cassette triggers high-efficiency multiplex genome editing in kiwifruit

Summary Kiwifruit is an important fruit crop; however, technologies for its functional genomic and molecular improvement are limited. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability is variable depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Optimizing conditions for its use within a particular species is therefore needed to achieve highly efficient genome editing. In this study, we developed a new cloning strategy for generating paired‐sgRNA/Cas9 vectors containing four sgRNAs targeting the kiwifruit phytoene desaturase gene (AcPDS). Comparing to the previous method of paired‐sgRNA cloning, our strategy only requires the synthesis of two gRNA‐containing primers which largely reduces the cost. We further compared efficiencies of paired‐sgRNA/Cas9 vectors containing different sgRNA expression devices, including both the polycistronic tRNA‐sgRNA cassette (PTG) and the traditional CRISPR expression cassette. We found the mutagenesis frequency of the PTG/Cas9 system was 10‐fold higher than that of the CRISPR/Cas9 system, coinciding with the relative expressions of sgRNAs in two different expression cassettes. In particular, we identified large chromosomal fragment deletions induced by the paired‐sgRNAs of the PTG/Cas9 system. Finally, as expected, we found both systems can successfully induce the albino phenotype of kiwifruit plantlets regenerated from the G418‐resistance callus lines. We conclude that the PTG/Cas9 system is a more powerful system than the traditional CRISPR/Cas9 system for kiwifruit genome editing, which provides valuable clues for optimizing CRISPR/Cas9 editing system in other plants.

Rapid radiations of both kiwifruit hybrid lineages and their parents shed light on a two-layer mode of species diversification

Reticulate speciation caused by interspecific hybridization is now recognized as an important mechanism in the creation of biological diversity. However, depicting the patterns of phylogenetic networks for lineages that have undergone interspecific gene flow is challenging. Here we sequenced 25 taxa representing natural diversity in the genus Actinidia with an average mapping depth of 26× on the reference genome to reconstruct their reticulate history. We found evidence, including significant gene tree discordance, cytonuclear conflicts, and changes in genome-wide heterozygosity across taxa, collectively supporting extensive reticulation in the genus. Furthermore, at least two separate parental species pairs were involved in the repeated origin of the hybrid lineages, in some of which a further phase of syngameon was triggered. On the basis of the elucidated hybridization relationships, we obtained a highly resolved backbone phylogeny consisting of taxa exhibiting no evidence of hybrid origin. The backbone taxa have distinct demographic histories and are the product of recent rounds of rapid radiations via sorting of ancestral variation under variable climatic and ecological conditions. Our results suggest a mode for consecutive plant diversification through two layers of radiations, consisting of the rapid evolution of backbone lineages and the formation of hybrid swarms derived from these lineages.

Whole transcriptome sequencing of Pseudomonas syringae pv. actinidiae- infected kiwifruit plants reveals species-specific interaction between long non-coding RNA and coding genes

An outbreak of kiwifruit bacterial canker disease caused by Pseudomonas syringae pv. actinidiae (Psa) beginning in 2008 caused disaster to the kiwifruit industry. However the mechanisms of interaction between kiwifruit and Psa are unknown. Long noncoding RNAs (lncRNAs) are known to regulate many biological processes, but comprehensive repertoires of kiwifruit lncRNAs and their effects on the interaction between kiwifruit and Psa are unknown. Here, based on in-depth transcriptomic analysis of four kiwifruit materials at three stages of infection with Psa, we identified 14,845 transcripts from 12,280 loci as putative lncRNAs. Hierarchical clustering analysis of differentially-expressed transcripts reveals that both protein-coding and lncRNA transcripts are expressed species-specifically. Comparing differentially-expressed transcripts from different species, variations in pattern-triggered immunity (PTI) were the main causes of species-specific responses to infection by Psa. Using weighted gene co-expression network analysis, we identified species-specific expressed key lncRNAs which were closely related to plant immune response and signal transduction. Our results illustrate that different kiwifruit species employ multiple different plant immunity layers to fight against Psa infection, which causes distinct responses. We also discovered that lncRNAs might affect kiwifruit responses to Psa infection, indicating that both protein-coding regions and noncoding regions can affect kiwifruit response to Psa infection.