Development of polyclonal antisera against movement proteins of the three poleroviruses infecting cucurbits and their serological relationships

Background: Cucurbit aphid-borne yellows virus (CABYV), Melon aphid-borne yellows virus (MABYV) and Suakwa aphid-borne yellows virus (SABYV) are three critical viruses infecting cucurbit crops. The preparation of specific antiserum against the virus is crucial for both the detection of virus and understanding the functions of the related genes. However, there is no report about detecting the three viruses using antisera against movement proteins (MP). Methods: In this study, we constructed prokaryotic expression vectors of the three viral movement proteins and transferred them into Escherichia coli strain Rosetta to purify the fusion proteins. Then the polyclonal antisera were obtained by immunizing New Zealand white rabbits. Western blotting was used to demonstrate the applicability of the three antisera. Results: We discovered that the titer of antiserum against MP CABYV reached to 1: 512000, and the titers of antisera against MP MABYV and MP SABYV reached to 1:256000. The optimized working concentration range for the three antisera was from 1:10000 to 1:64000. Both antisera against MP CABYV and MP MABYV could only react with the corresponding MP. The antiserum against MP SABYV not only had the strongest reaction with its MP but also could react with MP CABYV and MP MABYV at relative weaker levels and all the three antisera had no serological reactions with other poleroviruses tested. Furthermore, our results showed that the three antisera could specifically detect movement proteins both in Nicotiana benthamiana and cucumber leaves. Conclusions: We have established a sensitive system for detecting three poleroviruses infecting cucurbits by antisera against movement proteins, providing a material foundation for the future research on both the serological detection of viruses and the interaction mechanisms between the virus and host plants. MP: Movement ORF: Open PABYV: Pepo yellows virus; PLRV: Potato leafroll RT-PCR: Reverse SABYV: Suakwa yellows virus; ScYLV: Sugarcane virus; SDS-PAGE: Dodecyl Sulphate Gel TBST: Tris-buffered saline; ZABYV; Zucchini aphid-borne yellows virus.


Background
Cucurbitaceae is one of the most important fruits and vegetable crops in the world.
Interestingly, CABYV can also infect passion fruit and pepper naturally, which causes a large area of yellowing symptoms in fields [15][16][17]. MABYV and SABYV were first reported in China in 2008 and 2009, respectively [5,6]. MABYV is widely distributed in many provinces in China and Thailand; however, SABYV has been only detected in coastal provinces such as Fujian and Guangdong in southern China [2,6]. Subsequently, SABYV was reported in Southeast Asian countries such as Thailand, the Philippines, and East Timor [18][19][20]. All the three viruses are limited to the phloem tissues of the host plant and are transmitted by aphids in a persistent circulative and non-proliferative manner. They are highly specific in vectors and cannot be transmitted mechanically [21]. Symptoms such as yellowing and thickening of old leaves could cause severe yield reductions.
However, all three viruses have no significant effect on the quality and shape of fruits [4][5][6][7].
The virion of polerovirus including CABYV, MABYV and SABYV is a ball-shaped icosahedron with diameters of 25-30 nm, which encapsulate genome RNA. The virions are relatively stable and are not sensitive to chloroform and non-ionic detergents, but can be destroyed under high salt conditions with long time [22]. The genome consists of a single positivestranded RNA approximately 5.7 kb in length, containing seven open reading frames (ORFs). The first three ORFs of them are expressed by genomic RNA and the others are expressed by subgenomic RNA [23].Specifically, the P0 protein encoded by ORF0 is a classic RNA silencing suppressor, which can enhance the pathogenicity of viruses and can promote the accumulation of virus through interacted with host plants genes [24][25][26][27][28][29]. The P1 protein encoded by ORF1 serves as protease, helicase, and VPg. A P1-P2 fusion protein is encoded through frameshift and has a replicase activity [22]. The intergenic-non-coding region (intergenic-NCR) approximately 80 nt in length locates between ORF2 and ORF3a.
The P3a protein without AUG initiation encoded by ORF3a is indispensable for the longdistance movement of the virus [30][31]. The coat protein is encoded by ORF3, participating in the packaging, long-distance movement, and aphids transmission of the virus [32]. ORF4 encodes MP through leaky scanning, which could locate in plasmodesmata, mitochondria, and chloroplast to regulate much infection process including replication, cell to cell movement, and long-distance movement [33][34]. The read-through protein encoded by ORF5 is closely related to aphids transmission, virion assembly, long-distance movement, and phloem limited and so on [35][36][37][38].
Serological methods, as one of the essential methods for virus detection, are widely used due to its simple preparation, strong specificity, and high sensitivity. In the current research, RT-PCR is mainly used for the detection of CABYV, MABYV, and SABYV [18,39].
In regards to the serological detection, only antiserum against CABYV prepared by purified virion was once used in Western blotting or ELISA [4]. Some recent studies showed that polyclonal antiserum against MP can detect the accumulation of virus from the family Luteoviridae [40][41][42][43]. However, there are no reports on either preparing polyclonal antiserum against MP of the three viruses or the serological relationships among them. In our work, in order to obtain polyclonal antisera, we used His-MBP-MP purified fusion protein by prokaryotic expression system to immunize New Zealand rabbits. Western blotting showed that all the three viruses were detected by these antisera specifically. We Titer analysis of the three antisera We constructed MP CABYV , MP MABYV , and MP SABYV into the pMDC32 vector to express target proteins. Then we transiently expressed all the three MPs in N. benthamiana via agroinfiltration and the pMDC32 empty vector served as the negative control. Leaves were collected at three dpi to extract total proteins for western blotting, which showed that all the antisera could detect the corresponding MP respectively. Furthermore, antiserum against MP CABYV still could detect the specific band when the antiserum was diluted to 512000 times ( Fig. 2A). However, both antiserum against MP MABYV and antiserum against MP SABYV can detect the specific bands when these two antisera were diluted to 256000 times ( Fig. 2B and 2C). In summary, the titer of antiserum against MP CABYV reached to 1:512000 and both the titers of the other two antisera reach to 1:256000. From the perspective of color appearance and economics, the optimal working concentration range of the three antisera was from 1: 10000 to 1: 64000. identity in amino acid [43]. Among poleroviruses, Luffa aphid-borne yellows virus (LABYV), Pepo aphid-borne yellows virus (PABYV), and Cucumber aphid-borne yellows virus (CuABYV) also infect cucurbit crops [45]. In 2019, Zucchini aphid-borne yellows virus (ZABYV) was reported as a new virus species in China [46]. The MPs of these viruses have 23.08% -75.92% identity with the MPs of CABYV, MABYV, and SABYV in amino acid (Table 1). PABYV MP showed the highest homology, and LABYV MP showed the lowest homology among them. However, further experiments are indispensable to investigate the serological relationship between MPs of these viruses and the three antisera we developed.

The construction of vectors
The full-length of MP CABYV , MP MABYV , and MP SABYV were amplified by PCR using forward and reverse primers ( Table 2). The PCR products were recycled by 1.0% agarose gel electrophoresis and linked into the vector pDB.His.MBP (DNASU Plasmid Repository, Arizona, USA) digested with restriction enzymes NdeI and XhoI at 37℃ for more than 4 hours. We transformed the ligation products into E.coli strain MC1022 to obtain the positive clones and extracted plasmids. Sequencing analysis verified the correctness of plasmids. pMDC32-MP CABYV , pMDC32-MP MABYV and pMDC32-MP SABYV were constructed using the same methods [47].  Declarations manuscript.

Consent for publication
All the participants are consent for publication.