Analysis of subcellular localization and pathogenicity of Plum bark necrosis stem-pitting associated virus protein P6

Infection of plum bark necrosis stem pitting associated virus (PBNSPaV) has been reported in many prunus species in several countries, causing signi�cant economic losses. The very small proteins encoded by plant viruses are often overlooked due to their short sequences and uncertain signi�cance. However, numerous studies have indicated that they might play important roles in the pathogenesis of virus infection. The role of small hydrophobic protein P6, encoded by the open reading frame 2 of PBNSPaV, has not been well explored. In this study, we ampli�ed the P6 fragment from a PBNSPaV isolate by RT-PCR using speci�c primers and found that it is 174 bp long and encodes a protein of approximately 6.3 kD with a transmembrane domain. Subcellular localization analysis of P6 proteins in tobacco leaves showed that P6 localizes in cytomembrane and nuclear membrane. To further clarify the pathogenicity of P6 proteins, a PVX-P6 expression vector was constructed by inserting the p6 fragment into a potato virus X (PVX)-based vector and transformed into Agrobacterium tumefaciens GV3101. In�ltration of N. benthamiana with the PVX vector-transformed A. tumefaciens led to slight mosaic symptoms at 14 days of post-inoculation. Meanwhile, in�ltration with the PVX-P6 vector-transformed A. tumefaciens resulted in no signi�cant symptoms. These results demonstrated that heterologous expression of P6 in N. benthamiana could not enhance the pathogenicity of PVX. Our study indicates that p6 may not be a potential pathogenic factor associate with the causing of symptoms, and mode of action of PBNSPaV-P6 protein remain to be further studied.


Introduction
The stem-pitting disease was rst discovered in the 1960s in sweet cherry trees in North America.It has also been found in many Prunus species in many countries, including China [1][2][3][4].The disease is mainly caused by plum bark necrosis stem pitting-associated virus (PBNSPaV), which was rst reported infecting Japanese plum cv.Black Beaut in 1986 [5,6].Once infected, the trunk will secrete black gelatinous substances, the bark tissues will be severely necrotic, and stem pox symptoms will appear in the xylems, resulting in weak tree vigor, at branches, and other symptoms, which seriously endangers plant growth [5].China is one of the world's largest Prunus planting and producing countries [7,8].PBNSPaV infection was rst reported in China in 2011, causing signi cant economic losses [2,9,10].
However, studies on PBNSPaV are still lacking.
PBNSPaV is a non-enveloped, linear, single-stranded RNA virus belonging to the genus Ampelovirus of the family Closteroviridae [5].The complete PBNSPaV genome comprises seven major open reading frames (ORFs) with a total length of approximately 14 kb.Of the seven ORFs, ORF1a encodes a polyprotein, ORF3 encodes the heat shock protein (HSP) 70, ORF4 encodes a protein of about 61.6 kD, ORF5 and ORF6 encode coat proteins, and ORF2 encodes a 6.3-kD protein located between RdRp and HSP70 with unknown functions [5,6].Although the encoded proteins have been reported to be primarily involved in viral replication, motility, and mediator transmission [5,11], the functions of some PBNSPaV-encoded proteins, including P6, are yet to be determined.Especially, very small ORFs/proteins are often overlooked due to their short sequences and uncertain signi cance.Therefore, it is imperative to identify the coding regions and understand their functions to decipher how these small ORFs/proteins contribute to the infection cycle.To analyze the functions of P6 protein, the subcellular localization and pathogenic characteristics of P6 were studied by confocal microscopy and phenotypic observation, respectively.Our results laid a theoretical foundation for further analysis of P6's functions and mechanism in the interaction between PBNSPaV and its hosts.

Plant materials and bacterial strain
Sweet cherry leaves infected by PBNSPaV were collected from Shandong Province, in 2021.Fluorescent expression vector 35S:GFP, Agrobacterium GV3101, N. benthamiana seeds, and other plant materials were stored at our laboratory.PVX vector (pGR107) was a gift from Professor Xiaofei Cheng in Northeast Agricultural University [12] .

P6 Gene Cloning And Sequence Analysis
Total RNAs were extracted from 100 milligrams of cherry leaves using TRIzol reagent following the manufacturer's instructions and stored at -80℃.Primers used to amplify the P6 gene were designed based on the PBNSPaV sequence (GenBank: MZ221026).cDNAs were synthesized by rst incubating primers (Table S1), dNTPs, and total RNAs at 80℃ for 5 min, then with PrimeScript reverse transcriptase in 1×buffer (Takara Inc, Japan) at 42℃ for 1.5 h, and subjected to PCR for P6 gene ampli cation.The PCR product was cloned into the pMD18-T vector and transformed into DH5 competent cells.Positive clones were sequenced at Boshang Biotech Company (Jinan) for validation.The sequencing results were aligned to the PBNSPaV genome sequences on the NCBI database.Multiple alignments and homology analyses were performed using ClustalX and BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/), and the transmembrane prediction was performed using the TMHMM server (http://www.cbs.dtu.dk/services/TMHMM/).

Subcellular Localization Of P6
A P6-GFP plasmid was constructed by cloning the P6 gene into the 35S:GFP vector via BamHI and sequencing validated by Boshang Biotech Company (Jinan).The P6-GFP and GFP plasmids were transformed into Agrobacterium tumefaciens GV3101 using the CaCl 2 -mediated freeze-thaw method, as described previously.Single A. tumefaciens colonies were cultured in LB medium with 50 µg/mL kanamycin and 100 µg/mL rifampin at 28°C until OD600 reached 1.0−2.0.After centrifugation, the pellets were resuspended in the in ltration solution containing 10 mM MgCl 2 , 10 mM 2-(N-morpholino) and 0.15 mM acetosyringone to OD 600 of 1.2 and resuscitated at 28°C for 2 h.N. benthamiana at 5-7 leaf age was selected and treated on the back of leaves with resuscitated A. tumefaciens for 40-48 h using a 1 mL needless syringe.The in ltrated leaves were prepared as slides and observed under a laser confocal microscope (Zeiss, Germany) to check P6-GFP subcellular localization.The treatment was repeated by at least three times.

Pathogenicity Analysis Of P6
The P6 gene fragment was cloned via ligation using T4 ligase into the PVX expression vector, which was previously digested with Sal I and Cla I.The PVX heterologous expression vector and recombinant plasmid PVX-P6 were transformed into Agrobacterium GV3101 by freeze-thaw method and cultured on plates containing 50 mg/L kanamycin and 25 mg/L rifampicin at 28°C.Single colonies were selected for colony PCR identi cation.After identi cation, they were propagated overnight in liquid medium containing kanamycin and rifampicin to OD 600 reaching 0.6-0.8 and in ltrated into 5-7-leaf age N. benthamiana.After 7 and 14 days of in ltration, samples were collected, and their symptoms were recorded.In addition, the upper new leaves (non-invasive leaves) inoculated with PVX and PVX-P6 were fully ground after liquid nitrogen freezing and used to extract total RNAs.P6 and PVX expression were detected by RT-PCR using primer set P6-ClF and P6-SlR and primer set PVX-F and PVX-R, respectively.

Results
The obtained P6 gene is 174 bp long, encoding a protein of about 6.3 kD.BLAST with 24 reference isolates in GenBank showed that the P6 protein is highly similar to other reference proteins with only seven mutations (Fig. 1A).It indicated that P6 genes of PBNSPaV isolates are highly conservative in phylogeny.The P6 protein is a stable hydrophobic protein consisting of 57 amino acid residues and no signal peptide.It has an isoelectric point (pI) of 7.81 and structural analysis of the swiss model showed that the P6 protein mainly consists of -helix (Fig. 1C).Transmembrane prediction using TMHMM server (http://www.cbs.dtu.dk/services/TMHMM/)indicated that P6 is a transmembrane protein with transmembrane region composed of amino acid residues located at position 15 to 37 (Fig. 1B).
To determine P6 protein's localization in plant cells, the P6 sequence was fused in-frame to the 5' end of the GFP gene behind a 35S promoter to create p35S-P6:GFP vector.The expression vectors of GFP, P6-GFP, and CBL1-RFP (a membrane localization protein) were transformed into Agrobacterium GV3101, which were then used to in ltrate N. benthamiana.Confocal microscopic observation at 48 h of postin ltration revealed green uorescence signals in the cytomembrane and nuclear membrane of the epidermal cells of N. benthamiana in ltrated with P6-GFP and overlapped green and red uorescence signals excited by P6-GFP and CBL1, respectively.These observations indicated that P6 proteins are presented in both cytomembrane and nuclear membrane (Fig. 2A).To further study the pathogenicity of p6 proteins, expression vectors PVX and PVX-P6 were transformed into Agrobacterium GV3101, which were then inoculated into N. benthamiana, respectively.After inoculation with PVX-P6 for 14 days, the upper leaves of N. benthamiana showed no signi cant changes, while the upper leaves of N. benthamiana inoculated with PVX only showed slight mosaic symptoms (Fig. 2B).RT-PCR analysis of viral gene expression (primers in Table S1) showed that PVX was detected in all N. benthamiana inoculated with PVX and PVX-P6, while P6 was only detected in N. benthamiana inoculated with PVX-P6 (Fig. 2C).These results suggested that heterologous expression of P6 in N. benthamiana could not enhanced the pathogenicity of PVX.

Discussion
PBNSPaV belongs to the family Closteroviridae and possesses the largest and most complex genomes (up to 20 kb) of all positive-sense single-stranded RNA plant-infecting viruses.PBNSPaV is distributed worldwide, affecting a broad range of Prunus species [9,11].However, prevention and treatment of PBNSPaV infection are not well explored due to the lack of theoretical and practical studies.Therefore, analyzing the biological functions of viral proteins would contribute to understanding PBNSPaV infection mechanisms, the interactions of virus and plant hosts, and viral diseases prevention.In this study, we cloned the P6 gene of PBNSPaV from a sweet cherry isolate in Shandong Province and found that it is 174 bp long and encodes a protein of approximately 6.3 kD with a transmembrane domain.All P6 proteins have comparatively high similarity in amino acid sequences, and P6 genes of all PBNSPaV isolates are highly conservative in phylogenetic development (Fig. 1A).In addition to PBNSPaV, other members of the family Closteroviridae also encode small molecular weight proteins (P3.5, P6, and P8) [13][14][15][16][17].However, these proteins are different from the P6 protein in this study in coding position and homology with P6 in this study.Contrast to PBNSPaV-P6, the subcellular localization of other small proteins encoded by viruses in the family Closteroviridae might be different.For example, the P6 protein encoded by Cucurbit Chlorotic Yellows Virus (CCYV) distributes in the cytoplasm and nucleus [13], and P5 protein encoded by lettuce infectious yellows virus (LIYV) is an endoplasmic reticulum (ER) localized integral membrane protein capable of inducing ER stress [17].
Furthermore, several proteins of the same family could also induce necrosis in host plants and play important roles in viral infection, spread, and pathogenicity.PVX vector-mediated overexpression of P22, a viral gene silencing suppressor that plays an essential role during virus infection, causes plant necrosis [18].P5 and P9 proteins encoded by LIYV are produced in LIYV-infected plants and are essential for e cient viral infectivity [17].However, Our study indicates that p6 may not be a potential pathogenic factor associate with the causing of symptoms.These ndings suggest that these small proteins are similar in certain aspects, but they may function differently during virus infection, and the speci c function and mode of action of PBNSPaV-P6 protein remain to be further studied.

1
Abbreviations bark necrosis stem pitting associated virus PBNSPaV potato virus X PVX 0pen reading frames ORF