Molecular Characterization of Cylindrical Inclusion Protein Gene Regions of Turkish Zucchini yellow mosaic virus (ZYMV) Isolates Türkiye ZYMV İzolatlarının CI Protein Gen Bölgelerinin Moleküler Karakterizasyonu

: Zucchini yellow mosaic virus (ZYMV) is an economically important viral pathogen that causes intense mosaic symptoms and disfigurement in cucurbits. The aim of this study is to characterize ZYMV isolates obtained from Ankara, Antalya, Burdur, Konya, Karaman, Aksaray provinces of Turkey according to cylindrical inclusion (CI) protein sequences and to determine conserved areas on protein in Potyviruses. For this purpose, molecular studies and sequence analyzes were performed with primers specific to the CI protein region of collected cucurbit samples during 2019-2014 years. At the end of the study, the N terminus of Turkish ZYMV's CI protein 888 nucleotides long and 296 amino acids (aa) was amplified. Phylogenetic analysis of the nucleotide sequences of the CI region showed that the majority of isolates (40) belonged to a large molecular subgroup (A1) most common in Europe and the world, and three isolates (Y4, Y21, Y23) belonged to subgroup A5. Moreover, according to the coat protein nucleotide analysis, these three isolates (Y4, Y21, Y23) were grouped with the molecular subgroup A4, the group that emerged recently in Europe. The CI ZYMV nucleotide binding motif (NTBM) and RNA helicase activity site (five motifs) were conserved among isolates according to amino acid analysis. bu üç izolat (Y4, Y21, Y23), Avrupa'da son zamanlarda ortaya çıkan grup olan A4 moleküler alt grubu ile gruplanmıştır. CI ZYMV nükleotid bağlama motifi (NTBM) ve RNA helikaz aktivite bölgesinin (beş motif) aa analizine göre izolatlar arasında korunduğu görülmüştür.


Introduction
Zucchini yellow mosaic virus (ZYMV) causing intense mosaic symptoms and abnormalities in infected in cucurbit crops, causes significant economic losses and is included in the Potyviridae family, the Potyvirus genus (Choi et al., 2007;Desbiez & Lecoq, 1997;Hull, 2002). The genus Potyvirus is the largest genus of plant viruses. There are more than 200 species and more uncertain species in this group. Viruses belonging to this genus cause great economic losses by decreasing the quality and quantity of cucurbit products (melon, watermelon, cucumber, squash). The symptoms caused by ZYMV in plants are usually yellow mosaic on the leaves, severe deformation, swelling, significant shrinkage of the leaf blades, and severe stunting. In fruits, it causes swellings such as tubers and causes deformities on them. Infected melon and watermelon fruits also develop misshapen and longitudinal deep cracks. Seed formation is significantly reduced in infected plants and seeds are often deformed. In the tropic regions, ZYMV is often found as a complex with PRSV-W and WMV. Like other strains of Potyviruses, ZYMV is transmitted non-persistently by aphid species such as Aphis gossypii and Myzus persicae (Desbiez & Lecoq, 1997).
ZYMV particles are a 750 nm long, filamentous polyprotein consisting of a positive-sense RNA molecule (Hull, 2002;Lisa & Lecoq, 1984). At the 5th end of the genome, there is the genome linked protein (VPg) and at the 3rd end there is the polyA tail. The virus genome encodes 10 functional proteins (Glasa & Pittnerova, 2006;Revers et al., 1999, Urcuqui-Inchima et al., 2001. These are P1 protein, which acts as a protease in opening the genome, auxiliary component protein (helper component protein, HC-Pro), which plays a role in systemic transport of the virus by insects and suppresses gene silencing, cylindrical inclusion protein (CI) that plays a role in cell-to-cell transport and has RNA helicase activity; nuclear inclusion b protein (NIb), which plays a role in genome replication, and cover protein (CP), which plays a role in the systemic transport of the virus from cell to cell. CI plays a role in different stages of viral infection. It has been reported that the CI protein plays a role in virus replication, cellto-cell, and long-distance transport through interactions with the viral P3N-PIPO protein. CI protein acts as an avirulence factor in gene-for-gene interactions with dominant-resistance host genes and as a recessiveresistance overcoming factor (Sorel et al., 2014). Many viral and plant factors in plant cells have been shown to interact with this protein (Figure 1). In recent years, many studies on the biological and molecular variability of ZYMV have been published in the World (Coutts et al., 2011;Glasa et al., 2007;Maina et al., 2017;Novakova et al., 2014;Yakoubi et al., 2008). Most molecular studies have been based on the analysis of CP and/or partial NIb-CP sequences. It has also been reported that in the absence of the complete genomic sequence, the cylindrical inclusion (CI) coding region can be used for diagnostic and taxonomic purposes (Adams et al., 2005;Ha et al., 2008;Lee et al., 1997).

Material method 2.1. RNA isolation
In this study, total RNA isolation studies from samples (Tablo 1) collected from Konya, Karaman, Aksaray, Burdur, Ankara, and Antalya provinces were performed by Astruc et al. (1996) according to the method proposed.
RT-PCR was performed using RNAs obtained after total RNA isolation from samples taken from different provinces and different cucurbit plants. In the two-step RT-PCR process, firstly, cDNA was obtained from RNAs obtained from RNA isolation by using random hexamer primer and MMLV-RT enzyme. In the second step, these cDNAs were used as templates and PCR was carried out with primers synthesized specifically for CI protein regions.

Phylogenetic analysis
Protein regions of 43 plants from different provinces and districts that gave positive results at the end of RT-PCR were studied. The PCR products with positive bands at the end of the RT-PCR process were sent for DNA sequence analysis by Sanger method. The data obtained at the end of the sequencing were cleaned using the Chromas program and saved as a single file. Sequencing studies were carried out bidirectionally, and the obtained forward and reverse sequences were combined using the "CAP3 Sequence Assembly" computer program and consensus sequences were obtained. Based on the results obtained, the amplified parts of the isolates were compared with the isolates previously reported in the NCBI gene bank, and similarity rates were determined. For this purpose, the data were sequenced using the MEGAX (Kumar et al., 2018) program and a phylogenetic tree was created. Phylogenetic analysis was obtained using the "Neighbors joining tree" analysis method, 1000 boostrap and Kimura 2 parameters.

Results and Discussion
In the study, samples were taken collected from cucurbit plants showing virus symptoms from Ankara, Antalya, and surrounding provinces (Konya, Karaman, Aksaray, Burdur) where cucurbit cultivation is intensively performed. After the diagnosis of ZYMV positive isolates with serological tests (Topkaya et al., 2019), sequence analysis of the cylindrical inclusion protein gene regions encoded by the positive ZYMV isolates was performed, and phylogenetic analysis was performed by comparing the protein gene regions of the ZYMV isolates available in the literature.

RT-PCR results
In the amplification of the CI protein gene region of the ZYMV isolates used in the study, ZY-debCI-5' and ZY-milCI-3' primers, which amplify the partial region (N-terminal part) of the CI region (approximately 1000 bp), were used and at the end of the RT-PCR process, approximately 1000 bp bands were obtained in 1% agarose gel (Figure 2). GenBank. The CI nucleotide sequences were translated to amino acids using MEGAX computer software programs. Phylogenetic trees were done by neighborjoining (NJ) methods implemented in MEGAX (Kumar et al., 2018), with 1000 bootstrap replicates.
In the DAMBE program, isolates according to CI region, G1, 2, G3 isolates; BE13, BE15, BE18, BE22 isolates; BE5 and BE6 isolates; BE7, BE10, and C17 isolates; BRD1, BRD4 and YUN8-4 isolates; KZN1 and D14 isolates showed similar base sequences among themselves. While Y23 and Y4 in the A5 subgroup showed similar base sequences, Y21 showed a different sequence from these two isolates (Figure 3). The genetic distance between Turkish ZYMV isolates and with isolates from different parts of the world was 0.001-0.1 and genetic identity between groups was 90-99% (Table  2). Genetic identity indicated a very distant genetic relationship between the groups.

Figure 3: Phylogenetic tree of CI protein region Şekil 3. CI protein bölgesinin filogenetik ağacı
The isolates obtained in this study are shown in red, and the isolates from the GenBank database are shown in black. Boostrap values above 80% are shown.   The protein sequence generated using the nucleotide sequence of the CI protein region and the protein comparison with reference isolates were made using the MEGA6 computer program. The results obtained are shown in Table 1. The 296 amino acid partial protein sequence consisting of 888 nucleotides of the CI protein region was compared with reference isolates selected from available sequences in the NCBI gene bank. When the amino acid regions were evaluated at the end of the comparisons, it was found that the CI protein region was not very variable and the previously reported NTP binding motif "GAVGSGKST" (26-35. aa) and RNA helicase function "VLLLEPTRPL", "KVSAT", "LVYV" and "VATNIIENGVTL" motifs appear to be preserved (Table 3).
According to different researchers, the ZYMV CP region is divided into three main groups (Desbiez et al., 2002;Ha et al., 2008;Zhao et al., 2003). Firstly, ZYMV isolate were classified into two main groups based on the analyses of partial nt sequences of CP gene by Desbiez et al. (2002). Later Zhao et al. (2003) and Ha et al. (2008) reported three groups: I, worldwide; II, containing isolates only from Asia; and III, containing isolates only from China.
In previous studies, Lee et al. (1997) compared the CI protein gene regions of 14 ZYMV Singapore isolates and found five more conserved regions in addition to the sequence of the nucleotide binding (GAVGSGKST) motive, which is seen as the membrane binding component of RNA helicase complexes. In addition, as a result of the phylogenetic tree they made with the sequences of the CI and CP gene regions, similar branches were found, and they showed that the phylogenetic relationship between Potyviruses could be determined using the CI gene region. Researchers have suggested that the CI gene region can be used as an alternative approach in the evolution studies of Potyviruses.
The sequences of the (CP) and (CI) proteins of ZYMV isolates from Austria, Germany, Italy and Slovenia have been reported. As a result of DNA sequence comparison of 30 ZYMV isolates from different geographical regions around the world, it was seen that the Austrian isolates showed a high level of similarity with the Slovenian and Hungarian isolates. The isolates from Germany and Italy turned out to be distantly related and clustered with isolates from other parts of the world. The results of the study showed that a specific isolate can spread rapidly to geographically adjacent areas, but may not be related to isolates found in other neighboring countries (Pfosser & Baumann, 2002)

Conclusion
This is the first report of CI protein sequence information of the ZYMV in Turkey. Analysis with the CI region yielded results similar to the results obtained from the CP region and showed that the CI region could be used to classify ZYMV isolates. This study helps us to further understand the genetic diversity of ZYMV isolates infecting cucurbit plants collected from different provinces of Turkey.