Expression and variation of the genes involved in rhizobium nodulation in red clover

Authors: Dinkins, Randy; HANCOCK, JULIE - University Of Kentucky; Bickhart, Derek; Sullivan, Michael; ZHU, HONGYAN - University Of Kentucky

Submitted to: Plants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/25/2022
Publication Date: 11/28/2022
Citation: Dinkins, R.D., Hancock, J.A., Bickhart, D.M., Sullivan, M.L., Zhu, H. 2022. Expression and variation of the genes involved in rhizobium nodulation in red clover. Plants. 11(21). Article 2888. https://doi.org/10.3390/plants11212888.
DOI: https://doi.org/10.3390/plants11212888

Interpretive Summary: Red clover is an important forage crop and serves as a major contributor of nitrogen input in pasture settings because of its ability to fix atmospheric nitrogen. Red clover has not been a major contributor in legume/rhizobium studies primarily due to the issues associated with self-incompatibility and difficulties obtaining homozygous plants for genetic analysis compared to the model species currently used. Using recently developed red clover genomic resources and RNA-seq expression analysis, we describe the genes that are expressed in nodule forming roots. As has been observed for other species, red clover has many of the genes involved in rhizobium symbiosis that are conserved with those of other legumes, although some of the genes are red clover-specific with little or no homology with other legumes. These include the nodule-specific cysteine rich proteins (NCRs) while have common features, i.e. conserved cysteine residues, lack sequence homology and likely regulate the specific plant genotype/symbiont strain interactions. With the knowledge of the expression of these genes and genetic variation in red clover, breeding for increased nitrogen fixation efficiency by selection of genotypes with increased rhizobium specificity should be feasible.

Technical Abstract: Red clover (Trifolium pratense L.) is an important forage crop and serves as a major contributor of nitrogen input in pasture settings because of its ability to fix atmospheric nitrogen. During the legume-rhizobial symbiosis, the host plant undergoes a large number of gene expression changes, leading to development of root nodules that house the rhizobium bacteria as they are converted into nitrogen-fixing bacteroids. Many of the genes involved in symbiosis are conserved across legume species, while others are species-specific with little or no homology across species and likely regulate the specific plant genotype/symbiont strain interactions. Red clover has not been widely used for studying symbiotic nitrogen fixation, primarily due to its outcrossing nature, making genetic analysis rather complicated. With the addition of recent annotated genomic resources and use of RNA-seq tools, we annotated and characterized a number of genes that are expressed only in nodule forming roots. These genes include those encoding nodule-specific cysteine rich peptides (NCRs) and nodule-specific Polycystin-1, Lipoxygenase, Alpha toxic (PLAT) domain proteins (NPDs). Our results show that red clover encodes one of the highest number of NCRs and ATS3-like/NPDs, which are postulated to increase nitrogen fixation efficiency in the Inverted-Repeat Lacking Clade (IRLC) of legumes. Knowledge of the variation and expression of these genes in red clover will provide more insights into the function of these genes in regulating legume-rhizobial symbiosis and aid in breeding of red clover genotypes with increased nitrogen fixation efficiency. obium selection specificity and increased nitrogen fixation efficiency.