Conserved structure of the NPR1 gene distal promoter isolated from a chili pepper ( Capsicum annuum L.) in West Sumatera [version 2; peer review: 3 approved with reservations]

Background: The non-expressor of pathogenesis related gene 1 ( NPR1 ) protein is one of the key regulators in the systemic acquired resistance plant defense system. The cis -acting elements of its distal promoter gene are characterized by salicylic acid inducing elements such as the W-box, RAV1AAT and ASF1, accompanied by enhancer and silencer elements. This study was aimed to isolate and characterize the distal promoter sequence of the NPR1 gene ( PD_CbNPR1 ) from the chili pepper ( Capsicum annuum L.) genotype Berangkai , a local genotype known to produce large yields, but is susceptible to viral infection. Elucidating its sequence structure will open a broad range of possibilities to engineer the NPR1 gene expression which is important to improve chili pepper resistant. Methods: PCR-based cloning combined with a primer walking strategy was applied in this study. The BioEdit tool was used to edit the sequence and verify sequence integrity, while homology analysis was conducted with BLASTn searching. Identification of a cis -acting detected by PlantCare, and PlantPAN. complete distal promoter sequence of PD_CbNPR1 bp size. BLASTn search conserved (99% a single nucleotide polymorphism (SNP) (base compared its elements, which is characterized by a CCAAT-box and GAGAAATT pattern, respectively. Conclusion: The distal promoter of the NPR1 gene is highly conserved, showing only one SNP caused by one base substitution event. from each walking step was proc-essed to sequencing reaction using its both forward and reverse primers. All sequencing processes were performed by 1 st BASE-Singapore. A number of bioinformatic tools were applied during analysis of the sequence. Trimming, editing and building of the sequence contig were performed using BioEdit v7.2.5 (Hall, 1999). Homology search of the sequence with all available sequences deposited in the NCBI database was run using the BLASTn tool (Altschul et al ., 1990). The cis -acting elements were identified using PLACE, developed by et PlantCARE (Lescot et and PlantPAN. The authors cloned and sequenced the distal promoter region of the PD_CbNPR1 gene of chili pepper Berangkai, a genotype from West Sumatera that produces large yields but is prone to gemini virus infections. The sequence data was analyzed using several bioinformatics tools, and a number of cis-acting elements were identified. In the manuscript “Conserved structure of the NPR1 gene distal promoter isolated from a chili pepper (Capsicum annuum L.) in West Sumatera”, Dr. Jamsari Jamsari and co-authors offer us the structure of the distal promoter of the NPR1 gene, which will lay a foundation for the understanding of the mechanisms of pepper resistance to viral infection. But there are some issues that need to be explained:


Amendments from Version 1
Some changes in our 2nd version dealt with the comments and suggestions addressed by the two reviewers Minghui Lu, and Santy Peraza-Echeverria. In the Abstract: two bioinformatic tools: PlantCare and PlantPAN are added. The term "homology" has been changed to "identity". In the Methods: The "Genomic DNA isolation" subsection has been expanded. PlantCARE (Lescot et al., 2002) and PlantPAN are mentioned in the "Bioinformatic tools and sequence analysis" subsection. In the Results and Discussion: Figure 1 has been replaced and now shows the transcription start site and the length of core promoter.
In the "Characteristics of the NPR1 distal promoter" subsection, we add a paragraph explaining that PCR based cloning strategy in combination with primer walking was applied to isolate the complete full length of putative distal promoter NPR1 gene region (5,950 bp). The terminology "99% homology" was changed to "99% identity". We add that the BLAST analysis didn't show any significant homology with another promotor sequence available in the NCBI database, indicating a limitation of promoter sequence availability. We further expand on this and add a reference to Lee et al., 2006. We expand on our validation process and add a new reference (Nova et al., 2019). We have updated Figure 3 with 1 TCA motif and 3 CGTA motifs in the cis-acting diagram and the word "nine" is changed to "eleven". We added a new paragraph explaining the findings following analysis with PlantCare and PlantPAN 3.0. In the Conclusion: we add TCA and CGTA to the list of cis-acting elements and mention that the role of cis-acting elements in gene expressions needs to be confirmed.

REVISED
genotype is known to produce more yield, but susceptible to geminivirus infection. Resistant genotype however, is not available in our collection so far, so comparison of both two genotypes is not possible to be performed. The plant was grown in a greenhouse and maintained for 8 weeks before being used for DNA isolation. Genomic DNA isolation was performed using protocol as described by Jamsari & Pedri (2013).
Fragment isolation and PCR conditions PCR-based cloning combined with a primer walking approach was applied for the isolation complete segment of distal promoter of the NPR1 gene. All PCR reactions in this study were performed using the KOD-Plus-Neo kit provided by Toyobo-Japan. Chromosome 7 of Capsicum annuum cv. Zunla-1 (Qin et al., 2014), accession number: NC_029983.1, was used as the reference sequence. Primer combinations were designed to cover the interval segment spanning from base 112.600.897 to 112.606.847 on the reference sequence (Table 1). All primers used in this study are listed in Table 1. First-step isolation was started from both termini and continued with successive isolation until both termini formed a complete contig of the target segment. The initial PCR condition was started with 95°C for 3 minutes for pre-denaturation and amplified in two different loops using 14 and 24 cycles. The first 14 cycles were performed using a touch down steps started by denaturation at 95°C for 30 seconds. Annealing was initiated with 70°C for 30 seconds and gradually decreased 1°C for every cycle before finally elongated in 72°C for 2 minutes. The next 24 cycles started with denaturation at 95°C for 30 seconds with annealing at 55°C for 30 seconds and elongation of at 70°C for 2 minutes and elongated for 72°C for 2 minutes. The final extension was maintained at 72°C for 5 minutes. PCR reaction was performed in 50 µl of final volume. PCR composition was set according to manufacturer's recommendation, containing 5 µl 10x Buffer KOD-Plus-Neo, 5 µl of 2 mM dNTPs, 3 µl of 25 mM MgSO 4 , 1.5 µl of 10 ng/µl of each primer, 1 µl of KOD-Plus-Neo (Toyobo, Japan) and 1 µl of 10 ng/µl of DNA template. The volume was filled with 32 µl of nuclease-free water.

Bioinformatic tools and sequence analysis
The PCR product generated from each walking step was processed to sequencing reaction using its both forward and reverse primers. All sequencing processes were performed by 1 st BASE-Singapore. A number of bioinformatic tools were applied during analysis of the sequence. Trimming, editing and building of the sequence contig were performed using BioEdit v7.2.5 (Hall, 1999). Homology search of the sequence with all available sequences deposited in the NCBI database was run using the BLASTn tool (Altschul et al., 1990). The cis-acting elements were identified using PLACE, developed by Higo et al. (1999), PlantCARE (Lescot et al., 2002 and PlantPAN.

Introduction
The non-expressor of pathogenesis related gene 1 (NPR1) protein is the main regulator in the systemic acquired resistance response of many plants. Overexpression by modifying the distal promoter in the W-box element of the OsNPR1 gene in rice could increase its resistance against Xanthomonas oryzae pv. Oryzae up to 4.3 times (Hwang & Hwang, 2010). Similar results were reported by Zhong et al. (2015), who modified the RAV1 element on the GhNPR1 which increased Gladiolus hybridus resistance against Curvularia gladioli by up to 18.6 times compared to control. Based on those studies, we expect more prospects of the NPR1 gene promoter in the improvement of plant resistance against many pathogens.
Here we report characteristics of the distal promoter segment of the NPR1 gene isolated from chili pepper (Capsicum annuum) genotype Berangkai, a local genotype potentially produces more yields compared to other genotypes cultivated in West Sumatera.

Genomic DNA isolation
Healthy young leaves collected from chili pepper genotype Berangkai was used as the plant material in this study. This isolated from two steps round of walking via PCR-based cloning. In the first step, the whole distal promoter region was expected to be covered using two primer combinations (F1 F/R and F2 F/R). However, after verification of the sequence data using BioEdit, the upstream primer combination (F1 F/R) successfully produced a contig spanning only 1,963 bp, while the downstream primer combination (F2 F/R) was skipped for the sequencing process. In the second round of primer walking, four new primer combinations (F1.1-F/R, F1.2-F/R, F2.1-F/R, and F2.2-F/R) (Table 1) were designed in order to extend sequence coverage. Combining all verified sequences obtained from the first and the second round of primer walking successfully produced a contig with a size of 5,950 bp ( Figure 1). PCR based cloning strategy in combination with primer walking was applied to isolate the complete full length of putative distal promoter NPR1 gene region (5,950 bp). This approach is considered to be the most appropriate since the sequencing read capacity used in this study is limited for about only 500 bp on average. Even though the KOD-Plus-Neo could amplify up to 24 kb according to manufacturer's claim, but the full-length fragment (5,950 bp) still can not be sequenced in one step read due to limited reading capacity of the sequencing machine. Validation of every single nucleotide data was confirmed by at least two overlapping validated segment.
Homology analysis between PD_CbNPR1 and its reference sequence via BLAST search showed 99% identity. The data was verified by only one single nucleotide polymorphism (SNP) shown as a substitution event at the position -6,335 from the ATG start codon ( Figure 2). Furthermore, BLAST analysis showed no significant homology with another promotor sequence available in the NCBI database, indicating a limitation of promoter sequence availability. The only promoter sequence showed homology is the promoter region of Capsicum annuum pathogenesis related protein-1 (PR-1) gene (DQ201633.1) published by Lee et al. (2006). However, the comparable nucleotide of both sequences spanned only 180 bp.
The W-Box element binds the WRKY protein, acting as a transcription factor during expression of NPR1 (Yu et al., 2010). Mutation in this element in Arabidopsis thaliana delayed the NPR1 expression induced by salicylic acid (SA). PLACE analysis    indicated that PD-CbNPR1 is characterized by three consensus sequences: TTGAC, TGACY, and TGACT. The TGACY pattern could also be found in the promoter of ERF3 isolated from tobacco (Nishiuchi et al., 2004), while TGACT could be found in the promoter of the Iso1 barley gene (Sun et al., 2003). The WLE1 (W-box like elements) with TGACA pattern has an analog function with the W-box identified in the promoter of OsNPR1 (Hwang & Hwang, 2010).
Another cis-acting element found in PD_CbNPR1 is RAV1AAT, which acts as binding site for protein RAV1 (Hwang & Hwang, 2010). The RAV1 protein is a transcription factor associated with some pathogenesis-related genes (Sohn et al., 2006). Two consensus patterns of RAV1AAT elements found in PD_CbNPR1 are CAACA and TGTTG. The CAACA pattern was also found by Kagaya et al. (1999)  Another interesting element found in PD_CbNPR1 is the CAAT-box (26 repeats) which is associated with the regulation of many genes involved in pathogen infection (Imran et al., 2016). The CAAT-box consensus sequence found in PD_CbNPR1 is characterized with CAAT which is similar that have been found in promoter of the legA gene from Pisum sativum (Shirsat et al., 1989).
We also found a gibberellin-responsive element (GARE) motif, which has been previously reported by Ogawa et al. PLACE analysis also successfully identified four CCAAT-box motifs, which is commonly reported as an enhancer of transcription (Thonpho et al., 2013). This element is able to upregulate the transcription and replication rate of eukaryotic genes by binding many types of transcription factors (Biłas et al., 2016). da Silva et al. (2013) previously reported that a greater number of CCAAT-boxes could improve promoter capability in increasing of replication rate by binding efficiently with transcription factors. In contrast with the enhancer, we found only one motif of GAGAAATT which is also known as silencer (Lai et al. (2009). This element is reported to be able to down-regulate the kinesin-like protein 1 (AtKP1) gene in Arabidopsis thaliana by up to 80.9%.
Analysis with PlantCare (Lescot et al., 2002) resulting 2 new additional elements, which are designated TCA element and CGTA-motif (Figure 3), while analysis with PlantPAN 3.0 did not show any new different element compared to PLACE analysis. The TCA element has a function as a cis-acting element which is involved in salicylic acid responsiveness. Moreover we also found the CGTA motif playing a role in regulating me-JA responsiveness. In our sequence only 1 single TCA element and 3 CGTA motifs could be detected.

Conclusion
We successfully isolated the distal promoter of the NPR1 gene from chili pepper genotype Berangkai (designated PD_CbNPR1) spanning 5,950 bp. The PD_CbNPR1 contain 11 cis-acting elements: W-box, WLE1, RAV1AAT, TATA-box, CAAT-box, GARE, GT1, TCA, CGTA, enhancer and silencer elements. Engineering of the cis-acting elements may have future prospects, particularly in improving chili pepper resistance against biotic and abiotic stresses by up-and down-regulating NPR1 gene expression. However, their role in the gene expression has to be confirmed empirically. For that reason, we are currently using the cis-acting elements found in this study for replicase (Rep) gene expression in the bacterial system.

Data availability
The sequence of the distal promoter of the NPR1 gene sequenced in this study has been deposited in GenBank under accession number MK281381; https://identifiers.org/ena.embl:MK281381.

bipartite recognition sequence through two distinct DNA-binding domains
My biggest concern with this study is why focus on NPR1 to improve resistance to geminivirus infections? The examples they cited involve resistance to bacterial pathogens. The authors should cite examples of geminivirus resistance mechanisms that have been identified in other plants in their introduction and explain why NPR1 is a good target to enhance geminivirus resistance.
Another concern with this study is why did it focus on the distal promoter, and how was the distal promoter defined? In Figure 1 the authors define the core promoter as extending from -4050 to the Transcription Start Site. How was this defined? Has the Transcription Start Site been experimentally verified, and has the core promoter been analyzed elsewhere? I would expect to find most of the critical control elements within the core promoter, and these would be the most promising elements to engineer to enhance NPR1 expression.
A third concern is that it only compares this genotype with the source of the reference sequence. In order to use this sequence to provide insight into resistance to geminivirus infection they must compare it with resistant cultivars. In their Nova et al. (2019 1 ) paper they state that there are various chili pepper genotypes used in Indonesia. Are any of these more resistant to PepYLCV than Berangkai? Their statement that Berangkai produces large yields but is prone to geminivirus infection implies that other varieties are more resistant but produce lower yields.
In order to be useful for improving geminivirus resistance in chili pepper this study must identify sequences that vary between resistant and susceptible varieties which can then be tested experimentally. For author response 1, the authors can perform the seqmental sequencing using several primers inside the full-sequence.

References
For author response 2, the authors can find the homologus genes of NPR1 from other plant species, whose genomes were well-sequenced, like tomato, tobacco, even arabidopsis, and then get their distal promoter sequences based on the genome sequence.
For author response 3, only the sequence from a susceptible variety is not enough to explain its biological functions. I suggest the authors to compare the sequences from several varieties with different disease resistance (even a little difference).

Is the work clearly and accurately presented and does it cite the current literature? Partly
Is the study design appropriate and is the work technically sound? Partly

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility? Partly

Are the conclusions drawn adequately supported by the results? Partly
This study reports the cloning and bioinformatic analysis of the putative promoter region of PD_CbNPR1 from chili pepper. This putative promoter region was isolated from a local chili pepper genotype (Berangkai) known to produce large yields but susceptible to viral infections. The putative promoter sequence of PD_CbNPR1 could be useful for enhancing the immune system of the Berangkai genotype.
I have the following observations and comments: Indicate the Genbank accession number of the NPR1 sequence used to BLAST the chili pepper genome. Also, perform a phylogenetic analysis to determine whether the chili NPR1 sequence used to isolate PD_CbNPR1 belongs to the NPR1 clade (e.g., Arabidopsis has five NPR1 homologs: NPR2, NPR3, NPR4, NPR5 and NPR6. NPR1 and NPR2 form a single clade). 1.
Check the concept of homology. It is wrong to say "99% homology". 2.
The KOD-Plus-Neo kit is designed for long range PCR (up to 24 kb from human genomic DNA), so it is not clear why the amplification of the full-length putative promoter region (5950 bp single amplicon) was not possible.

3.
The search for putative cis-acting regulatory elements in the putative promoter of PD_CbNPR1 should also include other databases such as:  Figure 1: the length of the core promoter should be indicated along with the 5'UTR. 5.
The transcription start site should be provided as part of the characterization of the promoter region.

6.
A transient expression analysis using GUS as reporter gene would be useful to determine whether this putative promoter is functional.

If applicable, is the statistical analysis and its interpretation appropriate? Not applicable
Are all the source data underlying the results available to ensure full reproducibility? Partly
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