Functional Characterization Of A Mycorrhiza-Induced Transcription Factor OsHAP5G In The Arbuscular Mycorrhizal Symbiosis

Rice(Oryza sativa)is one of the most important food crops in China,and takes a central position in agricultural production,food security and national economy.Arbuscular mycorrhiza is a mutualistic symbiosis formed by the interactions between the arbuscular mycorrhizal(AM)fungi in soil and the roots of most land plants.With the exception of few plant species that have lost the capacity of forming AM symbiosis,most of the land plants worldwide,including a few of the early diverging plants from liverworts and ferns,can be colonized by AM fungi.In the AM symbiosis,AM fungi increase the transfer of mineral nutrients,in particular P and N,from the soil to plants;in return,they receive carbohydrates from the host plants for their proliferation.The establishment of AM symbiosis is a consequence of cooperative evolution between the two partners.Transcription factors are proteins that interact with cis-elements in the gene promoter region to activate or inhibit the transcription of target genes.It plays an important role in plant growth,morphogenesis and stress resistance.The formation of arbuscular mycorrhiza symbiosis is the result of a series of signal exchange and transduction,regulated by programmed expression of many genes.As a kind of important signal molecules,transcription factors have been proven to be able to participate in the regulation of the interaction between plants and AM fungi.In our preliminary experiment regarding RNA sequencing of the rice mycorrhizal and non-mycorrhizal roots,we found that many transcription factors,such as HAP,ERF and MYB,were substantially up-regulated or down-regulated in roots and/or leaves in response to AM symbiosis.Several HAP transcription factors have been demonstrated to be highly expressed in nodules of legume plants,with their roles having been proposed to be involved in the regulation of nodule development in legumes,however,it is not clear whether the mycorrhiza-regulated HAP transcription factors are required for the regulation of AM symbiosis.In this study,28 HAP/NF-Y/CBF family genes were identified by searching the whole rice genome.Quantitative expression of the genes in HAP5 subfamily of the HAP/NF-Y/CBF family revealed that OsHAP5G had the highest up-regulation induced by inoculation with AM fungi,suggesting this gene might be involved in the regulation of AM symbiosis.To test this hypothesis,we performed detailed investigations,such as subcellular localization,tissue-specific expression,as well as physiological analysis of the oshap5g knockout mutants and OsHAP5G overexpressing transgenic plants to dissect the potential roles of this AM-induced HAP transcription factor in the AM symbiosis.The main results were as follows:1.Bioinformatics analysis showed that OsHAP5G was located on the fourth chromosome of rice genome,with only one exon and no introns.The open reading frame of OsHAP5G is 378bp in length and encodes a protein consisting of 125 amino acids.Phylogenetic tree analysis showed that the HAP genes in rice were distributed in three subfamilies.Functional assay of OsHAP5G in yeast heterologous system showed that OsHAP5G has no transcriptional activation activity.2.Quantitative analysis of the OsHAP5G transcripts in different tissues showed that the OsHAP5G transcripts was weakly detectable in roots,leaves and leaf sheaths of non-mycorrhizal rice plants,but were strongly up-regulated in the roots colonized by the AM fungus(Rhizophagus irregularis).A time-course expression analysis revealed that the expression abundance of OsHAP5G was highly correlative to level of colonization intensity.Histochemical staining showed that the promoter of OsHAP5G could drive the GUS reporter gene strongly in the AM fungal-colonized roots,in particularly in the cells containing arbuscules.Subcellular localization analysis using the tobacco leaf transient expression system indicated that the protein encoded by OsHAP5G could be localized to the nucleus.3.In order to determine whether OsHAP5G is required for the regulation of AM symbiosis,homozygous oshap5g knockout mutants were generated with the CRISPR-Cas9 system using two different spacers targeting the coding sequence of OsHAP5G.After inoculation with the Rhizophagus irregularis for six weeks,the mycorrhizal infection rate were calculated.Our results showed that compared with wild-type(WT)plants,oshap5g mutants had a significant reduction in shoot biomass(dry weight),phosphorus(P)content and total colonization level as well as arbuscule incidence in mycorrhizal rice plants.4.To further investigate the potential role of OsHAP5G in AM symbiosis,we generated transgenic rice plants constitutively overexpressing OsHAP5G under the control of a maize ubiquitin promoter.After 6 weeks of growth inoculation with Rhizophagus irregularis,the total colonization level and arbuscule incidence in the OsHAP5G-Ox lines were significantly increased compared with those in the WT plants.Additionally,the expression level of the AM marker gene OsPT11,was also significantly increased compared with that in the WT plants.In conclusion,a mycorrhiza-induced transcription factor OsHAP5G was studied in this paper,through tissue expression pattern and subcellular localization characteristic analysis,as well as the physiological functions of mutants and overexpressed materials,it was made clear that OsHAP5G could be expressed in rice arbuscular cells after inoculation,and could be regulated rice mycorrhizal symbiosis process by controlling rice mycorrhizal infection and affecting the size and morphology of the arbuscular branches.