Identification Of The PtC/VIF Family And Their Expression Patterns In Response To Pathogenic Fungi In Poplar

In higher plants,invertase acts as the essential regulator in photosynthate partitioning,maintenance of sink strength,and stress response.C/VIF(cell wall/vacuolar inhibitor ofβ-fructosidase)is a family of small molecular weight proteins(14-23 k Da)that exist in the apoplast(cell wall)or vacuoles,inhibiting the hydrolysis activity of acid invertase depending on the post-translational mechanisms.C/VIF can also affect the development of plants and participate in the regulation of stress adaptation by adjusting the homeostasis of sucrose metabolism or cell osmotic pressure.However,C/VIF in woody plants remains unknown,and the genome of poplar has been published,which provides an opportunity to study the C/VIF gene of poplar.In this article,C/VIF gene members were identified and analyzed by bioinformatics.Tissue expression patterns of C/VIF and invertase genes and expression patterns under fungal stress conditions were studied by quantitative real-time PCR(q PCR)and a Pt C/VIF protein located in the apoplast was identified.The main results are summarized as follows:1.Based on the homologous search,a total of 39 members of the C/VIF gene family of Populus trichocarpa were obtained by BLAST search.The physical and chemical properties,signal peptide prediction,subcellular localization prediction,chromosome localization,gene structure,homology,conserved motif,and conserved domain of Pt C/VIF gene family were analyzed,showing the composition of 172-241 amino acid residues.The theoretical molecular weight of protein ranged from 18.42 to 26.60 k Da.All members were unevenly distributed on chromosomes 1 to 16.Promoter analysis showed multiple hormones and stress-related elements in the upstream of genes,including ABA,JA,and Me JA responsive elements and defense and stress-responsive elements.The phylogenetic tree showed that 39 C/VIF proteins and 13 reported C/VIF and PMEI proteins were divided into PMEI and C/VIF branches.2.RNA-seq and Microarray data showed that 12 and 10 genes were expressed in roots and leaves,respectively.A total of 6 genes were characterized by specific expression in the roots of tissue cultured Populus.Tissue-specific expression patterns of C/VIF and invertase genes(Pt CWI1 和 Pt VI2)under fungal stress(0h,24 h,48h,and72h)investigated are highly induced by q PCR assay.In general,the expression of most C/VIF genes in source tissues(leaves)was higher than that in sink tissues(roots and stems),indicating that C/VIF genes may be involved in the development regulation of P.trichocarpa.The response patterns of the C/VIF gene and invertase gene to Fusarium treatment were different.3.Bioinformatics and expression profiling revealed that Pt C/VIF1(potri.010g063000)had high identity to the reported homologous proteins in soybean and Arabidopsis,and specific expression in the roots tissue as well as responding to fungal infection.Therefore,we confirmed Pt C/VIF1 located apoplast by transient expression of tobacco leaves and stable transformation of Arabidopsis.The purified Pt C/VIF1 recombinant protein was co-cultured with CWI and VI to determine its specific inhibition of CWI.Finally,we confirmed that Pt C/VIF1 is a cell wall inhibitor of β-fructosidase.In summary,the identification of the Pt C/VIF family and the expression patterns in response to pathogenic fungi and functional characterization of Pt C/VIF1 provides a theoretical basis to unravel the physiological role and underlying regulatory mechanisms.These results pointed out that Pt C/VIF1 may exert potential functions in defense-related responses in poplar.