Comparative transcriptome analysis reveals compatible and recalcitrant genotypic response of barley microspore-derived embryogenic callus toward Agrobacterium infection

Abstract

Background: The Agrobacterium mediated transformation has been routinely used in lots of plant species as a powerful tool to deliver genes of interest into a host plant. However, the transformation of elite and commercially valuable cultivar is still limited by the genotype-dependency, and the efficiency of Agrobacterium infection efficiency is crucial for the success of transformation. Results: In this study, the microspore-derived embryogenic calli (MDEC) of barley elite cultivars and breeding lines were employed as unique subjects to characterize the genotypic response during Agrobacterium infection process. Our results identified compatible barley genotypes (GanPi 6 and L07, assigned as GP6-L07 group) and one recalcitrant genotype (Hong 99, assigned as H99) for the Agrobacterium strain LBA4404 infection using GUS assay. The accumulation trend of reactive oxygen species (ROS) was similar among genotypes across the time course. The results of RNA-seq depicted that the average expressional intensity of whole genomic genes was similar among barley genotypes during Agrobacterium infection. However, the numbers of differentially expressed genes (DEGs) exhibited significant expressional variation between GP6-L07 and H99 groups from 6 to 12 h post-inoculation (hpi). Gene ontology (GO) enrichment analysis revealed different regulation patterns for the predicted biological processes between the early (up-regulated DEGs overrepresented at 2 hpi) and late stages (down-regulated DEGs overrepresented from 6 to 24 hpi) of infection. KEGG analysis predicted 12 pathways during Agrobacterium infection. Among which one pathway related to pyruvate metabolism was enriched in GP6 and L07 at 6 hpi. Two pathways related to plant hormone signal transduction and DNA replication showed expressional variation between GP6-L07 and H99 at 24 hpi. It was further validated by qRT-PCR assay for seven candidate genes (Aldehyde dehydrogenase, SAUR, SAUR50, ARG7, Replication protein A, DNA helicase and DNA replication licensing factor) involved in the three pathways, which are all up-regulated in compatible while down-regulated in recalcitrant genotypes, suggesting the potential compatibility achieved at later stage for the growth of Agrobacterium infected cells. Conclusions: Our findings demonstrated the similarity and difference between compatible and recalcitrant genotypes of barley MDEC upon Agrobacterium infection. Seven candidate genes involved in pyruvate metabolism, hormonal signal transduction and DNA replication were identified, which advocates the genotypic dependency during Agrobacterium infection process.