Integrated analysis of plant gene expression and bacterial and protozoan community composition reveals changes related to root zonation, root cap and root hair formation

<p>This study was conducted within the framework of the DFG project SPP2089 “Rhizosphere Spatiotemporal Organization – a Key to Rhizosphere Functions” (project number 403641192).</p><p>As plant roots grow into the soil, the formation of biological gradients occurs at different spatial scales. It has been shown that plants recruit specific subsets of the soil bacterial community at their roots through excretion of mucilage at root tips and exudates at the sites of root hair formation. The promotion of or defense against certain bacterial taxa is also reflected in the composition of the protist communities that feed on bacteria.</p><p>Using high-throughput sequencing methods, we investigated emerging patterns in root gene expression in relation to bacterial and protozoan community structures. We found highly distinct root region specific patterns relating to differential root gene expression relating to growth, defense and transporter activity, as well as bacterial and protist (cercozoan) diversity. Root cap removal led to differently composed microbial communities, as well as a regulation of root genes relating to stress and defense. The lack of root hairs was only reflected in the amount of microbial carbon in soil and a small number of differentially expressed genes involved in cell wall processes.</p><p>We could show that the rhizosphere microbiome, is as dynamic as its environment. Root regions differentially affect microbial communities, which is also reflected in the expression of plant genes of categories relating to defense, immunity and stress. Our findings will further enhance our understanding of microbial root interactions at single root scale.</p>

Identifying and validating housekeeping hybrid Prunus sp. genes for root gene-expression studies

Prunus rootstock belonging to subgenera Amygdalus (peach), Prunus (plum) and Cerasus (cherry) are either from the same species as the scion or another one. The number of inter-species (including inter-subgenera) hybrids have increased as a result of efforts to broaden the genetic basis for biotic and abiotic resistance/tolerance. Identifying genes associated with important traits and responses requires expression analysis. Relative quantification is the simplest and most popular alternative, which requires reference genes (housekeeping) to normalize RT-qPCR data. However, there is a scarcity of validated housekeeping genes for hybrid Prunus rootstock species. This research aims to increase the number of housekeeping genes suitable for Prunus rootstock expression analysis.Twenty-one candidate housekeeping genes were pre-selected from previous RNAseq data that compared the response of root transcriptomes of two rootstocks subgenera to hypoxia treatment, ‘Mariana 2624’ (P. cerasifera Ehrh.× P. munsoniana W. Wight & Hedrick), and ‘Mazzard F12/1’ (P. avium L.). Representing groups of low, intermediate or high levels of expression, the genes were assayed by RT-qPCR at 72 hours of hypoxia treatment and analyzed with NormFinder software. A sub-set of seven housekeeping genes that presented the highest level of stability were selected, two with low levels of expression (Unknown 3, Unknown 7) and five with medium levels (GTB 1, TUA 3, ATPase P, PRT 6, RP II). The stability of these genes was evaluated under different stress conditions, cold and heat with the hybrid ‘Mariana 2624’ and N nutrition with the hybrids ‘Colt’ (P. avium × P. pseudocerasus Lindl.) and ‘Garnem’ [P. dulcis Mill.× (P. persica L.× P. davidiana Carr.)]. The algorithms of geNorm and BestKeeper software also were used to analyze the performance of these genes as housekeepers.Stability rankings varied according to treatments, genotypes and the software for evaluation, but the gene GBT 1 often had the highest ranking. However, most of the genes are suitable depending on the stressor and/or genotype to be evaluated. No optimal number of reference genes could be determined with geNorm software when all conditions and genotypes were considered. These results strongly suggest that relative RT-qPCR should be analyzed separately with their respective best housekeeper according to the treatment and/or genotypes in Prunus spp rootstocks.