Study on the interaction mechanism between Crocus sativus and Fusarium oxysporum based on dual RNA-seq

The saffron phenylpropane synthesis pathway and Fusarium oxysporum cell wall-degrading enzymes play key roles in their early interactions. Saffron (Crocus sativus) is a highly important crop with diverse medicinal properties. F. oxysporum is a widely-distributed soil-borne fungus, causing the serious saffron rot disease. Currently, there is no effective management strategy to control this disease because of no resistant cultivars and limited information about the resistance and pathogenic mechanisms. In this study, we first characterized the infection process and physiological responses of saffron infected by F. oxysporum. The molecular mechanism of these infection interactions was revealed by dual RNA-seq analysis. On the 3rd day of infection, the hyphae completely entered, colonized and spread in the corm cells; while on the 6th day of infection, hyphae had appeared in the xylem cells, blocking these vessels. Transcriptome results indicate that within the host, phenylpropanoid metabolism, plant hormone signal transduction and plant pathogen interaction pathways were activated during infection. These pathways were conducive to the enhancement of cell wall, the occurrence of hypersensitivity, and the accumulation of various antibacterial proteins and phytoantitoxins. Meanwhile, in the fungus, many up-regulated genes were related to F. oxysporum cell wall degrading enzymes, toxin synthesis and pathogenicity gene, showing its strong pathogenicity. This study provides new ideas for the control of saffron corm rot, and also provides a theoretical basis for mining the key functional genes.


Introduction
Crocus sativus L., also known as saffron, is a perennial bulbous herb of the genus Crocus in the Iridaceae family. Saffron is a native of Southern Europe and today cultivated worldwide in many countries, particularly in Spain, Italy, Iran and other countries of the Mediterranean area. In China, it is mainly distributed in Zhejiang, Beijing, Shanghai, Jiangsu. Saffron is not only a precious flavoring agent, high-grade dye, spice, but also a highly important medicinal plant. It has positive effects on improving immunity (Syed et al. 2018), anti-tumor (Colapietro et al. 2019) and treating cardiovascular diseases (Ghajar et al. 2017;Makan et al. 2019;Zahra et al. 2018). However, as only the stigma of saffron can be used for medicine and the yield is extremely low, it is also called "plant gold". China's saffron cultivation area is about 2000 hectares, and the annual output of stigmas is about 20 tons, which is far from meeting the market demand.
Saffron is a triploid sterile species that can only reproduce asexually through corms (Baba et al. 2015;Schmidt et al. 2019). In China, the cultivation of saffron adopts a two-stage method, namely indoor (June to October) storage and flower collection and outdoor (November to May) corm cultivation. Planting, digging and routine debudding operations usually cause damage to the corms and make them more susceptible to rot disease. In recent years, corm rot has become the most common and serious disease in saffron production, which not only directly reduces the quality and yield of stigma, but also affects the production of subcorms (Kour et al. 2018). Rot diseases are caused by a complex of several pathogenic microorganisms, with F. oxysporum as the most important (Di Primo and Cappelli 2000). F. oxysporum is a widely-distributed soil-borne pathogenic fungus that causes roots and corms rot in hundreds of cultivated plants, resulting in significant economic losses (Taylor et al. 2019). F. oxysporum mainly destroys the vascular tissue of plants and affects the growth and development of host plants by producing metabolites such as enzymes, phenols and toxins (Gordon 2017). After the plant dies, the fungus spreads to the plant surface, produces spores and disperses them into the soil for secondary infection (Bani et al. 2018). Compared with other Fusarium fungi, F. oxysporum has a high degree of host specificity (Ma et al. 2013). At present, the interaction mechanism between saffron and F. oxysporum is still unclear.
RNA-Seq technology has been used to study plant diseases. It can dynamically detect the spatiotemporal changes of plant gene expression of disease infection, as well as mining functional genes and analyzing the regulatory mechanism of host and pathogen interactions. The interactive transcriptome developed on this basis can be used to study the transcripts of the host and the pathogen at the same time, providing a more complete view of the interaction for finding the differentially expressed genes and host-pathogen interaction mechanism (Naidoo et al. 2018). Plant diseases are the result of host-pathogen interaction. Therefore, this study analyzed the interaction mechanism between saffron and F. oxysporum at the transcription level. Dual RNA-Seq was used to reveal the transcripts of plants and fungi at different time periods after infection, and the defense response of saffron and the pathogenic mechanism of F. oxysporum were analyzed. The research results provide new insights into the interaction mechanism between saffron and F. oxysporum, and also provide new ideas for the control of saffron corm rot.

Materials
Saffron corms were purchased from Jiande, Zhejiang province, and stored indoors in the shade. Fungi were isolated from rotten saffron corms and purified by single spore culture. According to morphology and rDNA-ITS sequence analysis, F. oxysporum was proved to be the most pathogenic fungus (Zhou et al. 2015).

Preparation of spore suspension and inoculation of corms
Healthy saffron corms of the current year were taken, each of which weighs about 20 g. After peeling off the outer membrane, the corms were rinsed by water for 4 h. The cleaned corms were subsequently put into the ultra-clean workbench, disinfected with 2% sodium hypochlorite solution for 30 min, washed with sterile water for three times, and dried for inoculation.
F. oxysporum was inoculated on a PDA medium and incubated at 28 °C for 15 days. Five pieces of 6 mm-size cakes were punched from the edge of the colony and then placed in the 50 mL PDB medium and incubated in a shaker at 28 °C. After 4 days, the spore suspension was obtained after filtering off the mycelium and counted under a microscope using a hemocytometer. The spore concentration was diluted to 10 6 spores/mL with sterile water for later use.
The corms were inoculated by puncture. The wounds were located at the 4th stem node ring under the terminal bud of the corm, about 0.5 cm deep. Three wounds per corm were inoculated with a total of 10 uL spore suspension. The corms of control group were inoculated with 10 uL sterile water. Two layers of filter paper were laid in the sterile culture bottle. After wetting the filter paper with 3 mL of sterile water, the inoculated corms were placed in a bottle and cultured in a constant temperature incubator at 28 ℃.

Microscopic observation on the process of F. oxysporum infecting saffron corms
At 0-7 days after inoculation, tissue blocks with a size of 0.5 cm 3 at the junction of saffron corm were collected and placed in formaldehyde-acetic acid-ethanol fixative solution and fixed overnight in a refrigerator at 4 ℃. The tissue blocks were made into slices with a thickness of 6 μm, which were prepared by dehydration, transparent, waxing, embedding, slicing, spreading, sticking, drying, dewaxing, solid green staining and sealing, and then observed and photographed under an optical microscope.

Sample collection and RNA-Seq analysis
Tissue pieces about 0.5 cm 3 in size were taken from the lesions of the saffron corms at 3rd and 6th day after inoculation, respectively. These two groups were interaction samples, which contain both saffron corms and F. oxysporum. Saffron corms inoculated with sterile water and F. oxysporum in the culture medium were used as control groups, respectively. Each group was repeated three times with a total of 12 samples. The total RNA was extracted and mRNA was enriched by Oligo (dT) beads. Then the enriched mRNA was fragmented into short fragments and reverse transcripted into cDNA. Second-strand cDNA were synthesized and the cDNA fragments were purified, end repaired, poly (A) added, and ligated to Illumina sequencing adapters. After size selected and PCR amplified, the products were sequenced in the Illumina Novaseq 6000.
The transcriptome sequencing clean reads were obtained after removing splice sequences and low-quality sequences. F. oxysporum has a high-quality reference genome, while saffron currently has no reference genome. Thus, clean reads were first aligned to the F. oxysporum reference genome using HISAT2 software (Ma et al. 2010). After removing F. oxysporum reads, the remaining reads were assembled as saffron transcripts using Trinity software (Grabherr et al. 2011). All the assembled unigenes were blastx to multiple nucleic acid and protein public databases, including NR, SwissProt, KEGG and COG/KOG, and the annotation results were aggregated as saffron unigenes data. For each transcription region, a FPKM (fragment per kilobase of transcript per million mapped reads) value was calculated to quantify its expression abundance and variations, using StringTie software. Differentially expressed genes (DEGs) were screened by FDR < 0.05 and |log2FC|> 1 according to DeSeq2 method. The Gene Ontology (GO) enrichment analysis of the DEGs was implemented by the GOseq R package (1.10.0) (Young et al. 2010). Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to predict signaling pathways of DEGs using KOBAS software (Mao et al. 2005). Plant TF database was used to predict saffron transcription factors and the PRG database was used to annotate saffron R genes. The carbohydrate active enzyme database was used to predict the carbohydrate enzyme of F. oxysporum, and the PHI-Base database was used to analyze pathogenic genes of F. oxysporum. The raw RNA-seq reads obtained in the study were submitted to the SRA database with the accession number PRJNA866873. The sequences of each unigene and the mean expression level of all genes were listed as supplemental files (Table. S1, S2).

QRT-PCR analysis
The extracted high-quality RNA was used as template and cDNA was obtained by reverse transcription using Evo M-MLV RT Premix kit (Agbio, Hunan, China). The qRT-PCR 20 µL reaction system was constructed using the SYBR Master Mix (Agbio, Hunan, China). Saffron 18SrRNA was used as the reference gene. All primers used in this study are listed in Table S3. Real-time assays were performed with SYBR Green Dye using CFX96 Touch ™ RealTime PCR (Bio-Rad, USA) detecting platform. The relative expression level of genes was calculated by the 2 − ΔΔCt method. Three biological replicates and technical duplication were used in RT-qPCR analysis.

Statistical analysis
All data represent the means of no less than three measurements, and results were reported as the mean ± SD (standard deviation). Significant differences were calculated by the One-way ANOVA and Student's t-test using GraphPad Prism 9.2.0 (GraphPad Software Inc., San Diego, CA, USA). P < 0.05 was considered statistically significant.

Phenotypic observation of saffron corms after F. oxysporum infection
At the 1st day after inoculation, a small number of hyphae appeared near the wound, while there was no significant change on the surface of the corm. After inoculation for 3 days, the color of the inoculation site became darker and the number of white hyphae increased. At the 7th day after inoculation, brown rotten symptoms were evident at the inoculation site, and F. oxysporum had proliferated and spread within the corm (Fig. 1A). At the 3rd day after inoculation, the internal region of saffron corm gradually turned yellowish brown (Fig. 1B). There was a significant difference in the diameter of the lesions at the 12th day after inoculation, at which point the saffron corms had been severely damaged (Fig. 1C).

Microscopic observation of saffron corms infected by F. oxysporum
In the control group inoculated with sterile water, no mycelia were observed and the corm cells remained intact, mostly round or oval ( Fig. 2A). At 12 h after inoculation, there were a few scattered hyphae around the inoculation site ( Fig. 2B). At the 2nd day after inoculation, the number of hyphae increased, the hyphae began to form clusters, interwining with each other, and a large number of conidia were distributed around the hyphae (Fig. 2D). At the 3rd day after inoculation, the hyphae were found to invade into healthy cells around the inoculation site and colonized and extended in the cells (Fig. 2E). At the same time, the hyphae aggregated along the intercellular space of the corm, and many hyphae penetrated the corm cells through the cell wall, causing cell damage (Fig. 2F, G). At the 6th day after inoculation, the cells were filled with hyphae, and they continued to expand to the inner cells of the corm, and conidia and hyphae were also found in the xylem cells (Fig. 2H, I).

Activities of resistance-related enzymes in saffron corm infected by F. oxysporum
The activities of CAT and POD in corm cells increased after F. oxysporum infection. CAT enzymes showed significant differences at the 1st day after infection, whereas POD enzymes peaked at the 3rd day, and then decreased. In the control group inoculated with sterile water, the activities of both enzymes also fluctuated, possibly due to mechanical damage caused by the punctures (Fig. 3A, B). The chitinase activity increased rapidly at the early stage of infection, and then dropped quickly after four days of infection. At the 7th day, the chitinase activity level was close to the control group (Fig. 3C). Compared with the control group, β-1,3 glucanase was only significantly different at the 5th day after infection, and the change was not obvious at the rest of the time (Fig. 3D).

Dual RNA-Seq analysis
After data filtering, a total of 553.19 M clean reads were obtained by dual RNA-Seq (Table. S4). For F. oxysporum, about 82% of the reads in the control group could be compared to the reference genome, and 2.5% of the reads in the interaction samples could be aligned to the reference genome (Table. S5). At the 3th day after infection, a total of 2644 differentially expressed genes were screened in F. oxysporum, including 1637 up-regulated genes and 1007 down-regulated genes, and at the 6th day after infection, a total of 2690 differentially expressed genes were screened, including 1680 up-regulated genes and 1010 down-regulated genes (Fig. 4C). The KEGG enrichment results of the transcriptomes of F. oxysporum showed that these differentially expressed genes were mainly related to the mutual transformation pathway of pentose and glucuronic acid, galactose metabolism, ABC transporter, sphinolipid metabolism, starch and sucrose metabolism (Fig. 4D).
The assembly of saffron transcriptome data yielded 84,544 Unigenes with an N50 length of 1561 bp (Table. S6). A total of 39,962 unigenes were annotated. At the 3th day after inoculation, 14,263 differentially expressed genes were screened, including 6835 up-regulated genes and 7428 down-regulated genes. Meanwhile, at the 6th day after inoculation, 16,397 differentially expressed genes were screened, including 7328 up-regulated genes and 9069 down-regulated genes (Fig. 4A). KEGG enrichment of differentially expressed genes showed that in the CK-vs-3dpi (3 days postinoculation) and CK-vs-6dpi (6 days post-inoculation) treatment groups, the significantly enriched pathways of saffron included metabolic pathway, secondary metabolite synthesis, phenylpropanoid biosynthesis, flavonoid biosynthesis, MAPK signal transduction, plant hormone signal transduction, etc. (Fig. 4B).

Analysis of phenylpropane metabolic pathway in saffron
The saffron phenylpropane metabolic pathway was significantly enriched in response to F. oxysporum infection. In this pathway, a total of 115 genes were differentially expressed. Phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-coumaric acid CoA ligase (4CL), as the three key enzymes in the phenylpropane pathway, catalyze the conversion of phenylalanine to p-coumaric acid and p-coumaryl CoA, which are the precursors of lignin synthesis. The expression levels of 16 PAL genes were significantly up-regulated, and 15 of them continued to increase with the prolongation of infection time. In addition, the expression level of C4H genes and 4CL genes also consistently increased. The expression of other lignin synthesis-related genes such as cinnamoyl CoA reductase (CCR), hydroxycinnamoyl transferase (HCT), caffeoyl CoA-O-methyltransferase (CCoAMT), caffeic acid-Omethyltransferase (COMT), and cinnamyl alcohol dehydrogenase (CAD) were also mostly consistently up-regulated with the increase of infection time. Peroxidase (POD) is one of the key enzymes for lignin synthesis. Among the 28 POD-related genes screened, except for three genes that were down-regulated compared with the control group, the others were all up-regulated. In the coumarin biosynthesis pathway, the expression level of 14 β-glucosidase (BGLU) genes was up-regulated, and in the scopoletin biosynthesis pathway, the expression level of the other TOGT1 genes increased except for two genes (Fig. 5).

Saffron hormone regulation process
For the salicylic acid (SA) signaling pathway, the regulatory protein gene NPR1 acts as a key gene that interacts with the transcription factor TGA to activate the expression of related genes and produce PR1 protein to enable plants to acquire disease resistance (Zhang and Li 2019). After F. oxysporum infection, the expression levels of all NPR1 genes in saffron increased continuously at 3dpi and 6dpi; the expression levels of three transcription factor TGAs were significantly up-regulated at the same time. The expression of PR1 downstream of salicylic acid signal transduction pathway was also significantly increased (Fig. 6A). For the jasmonic acid (JA) signaling pathway, jasmonic acid amino synthase 1 (JAR1) gene was upregulated continuously, whereas jasmonic acid ZIM structural protein (JAZ) genes were downregulated continuously from 3 to 6 dpi. JAZ is a negative regulator of JA pathway, and saffron promotes the jasmonic acid signaling pathway by down-regulating JAZ to improve its disease resistance (Fig. 6B).

Genes related to the plant-pathogen interaction pathway in saffron
The expression of CaM in saffron was significantly downregulated after F. oxysporum infection, while most of the genes encoding CDPK and Rboh were significantly up-regulated at 3dpi, which could cause cell wall enhancement and hypersensitivity reaction after a series of reactions in saffron (Fig. 7A). In addition, other genes involved in PAMP-triggered immunity (PTI), such as PR1, were continuously upregulated after infection, while transcriptional activators of pathogen-related genes, PTI5, PTI6 and PTI1, were significantly down-regulated (Fig. 7B). RIN4 is the target of many Fig. 3 Changes of enzyme activities related to resistance in saffron corms after F. oxysporum infection. A CAT activity, B POD activity, C chitinase activity, D β-1,3-glucanase activity (*p < 0.05, **p < 0.01, compared with the control group at the same time point) pathogenic effector proteins, one of the two RIN4 genes was up-regulated and the other was down-regulated. The RPM1 gene was up-regulated, while most of RPS2 showed a downregulated trend. In addition, the expression levels of HSP90 protein-related genes were mostly down-regulated (Fig. 7C).
At the 3rd day after inoculation, we observed 298 R genes were differentially expressed in saffron, with 177 were upregulated and 121 were down-regulated (Fig. 8C). At the 6th day after inoculation, we observed 356 R genes were differentially expressed in saffron, with 229 were up-regulated and 127 were down-regulated (Fig. 8D). The R genes differentially expressed in the two infection periods were of the same type, including KIN, RLK, RLP, N and other 13 types. Among these R genes, the number of KIN genes was the largest.

Confirmation of the transcriptome data using qRT-PCR
A few defense related genes with differential expression in saffron were randomly selected and validated by qPCR analysis. The results showed that all gene expression trends were consistent with the transcriptome sequencing results except for Unigene0078857 (MPK4) (Fig. 9). It is speculated that this may be caused by the difference between the used two methods.

Analysis of pathogenic genes related to F. oxysporum
Polygalacturonase and β-glucosidase are the main cell wall degrading enzymes secreted by F. oxysporum (Bravo et al. 2016;Olajuyigbe et al. 2016). During F. oxysporum infection, the gene expression levels of two endo-polygalacturonases PG1 and PG5 were increased by 9.6 and 5.2 times, respectively. The gene expression levels of two exdo-polygalacturonases PGX1 and PGX4 were increased by 8.8 and 14.4 times, respectively. Meanwhile, the gene expression levels of β-glucosidase were increased 6.9 and 2.9 times, respectively. In addition, xylanase gene expression was also significantly up-regulated (Table 1).
The pathogenic genes of F. oxysporum were predicted in the PHI database, and two genes related to the growth and development of F.oxysporum were found. Among them, the REN1 controls the development of small and large conidia of F. oxysporum (Ohara et al. 2004), while CHS2 is associated with cell wall genesis (Martin-Udiroz et al. 2004). Both of them decreased in expression during infection. In addition, we observed 16 genes related to the virulence of F. oxysporum. FGB1 and Fmk1, as key genes of MAPK signaling pathway, increased significantly during the infection process. Histone kinase Fhk1 controls stress adaptation and virulence of F. oxysporum (Rispail and Di Pietro 2010), and its expression level increased. Decreased expression of the PH-signaling transcription factor PacC, a negative regulator of virulence, prevents transcription of acid-expressing genes related to F. oxysporum infection (Caracuel et al. 2003). Protein kinase SNF1 is associated with transcription of cell wall degrading enzymes (Ospina-Giraldo et al. 2003) and its expression level was continuously up-regulated. The expression levels of two zinc finger transcription factors FOW2 and XLNR increased, which controlled the expression of pathogenic genes involved in F. oxysporum. Five lipase genes were identified, all of which were up-regulated in expression during infection. They may be related to the vegetative growth of F. oxysporum (Bravo-Ruiz et al. 2013).   The expression of Gas1 encoding β-1, 3 glucanyl transferase decreased. The expression of ARG1, which encodes arginine succinate lyase and catalyzes the final step of arginine biosynthesis, was increased, and ARG1-deficient strains showed reduced pathogenicity (Namiki et al. 2001) ( Table 2).

Analysis of genes related to toxin synthesis in F. oxysporum
Fusaric acid synthesis-related genes FUB8, FUB10, FUB11, FUB12 had very high basal expression levels when F. oxysporum was grown in the medium, but the expression levels were significantly reduced after inoculation into saffron corms (Fig. 10A). In addition to fusaric acid, the trichothecenes-related genes TRI8, TRI13, and TRI14 were differentially expressed, and the expression levels of these three genes were significantly increased (Fig. 10B). A total of seven fumonisin-related genes, FUM2, FUM6, FUM7, FUM15, FUM17, FUM18, and FUM19, were identified in the transcriptome data, and all of them were up-regulated except FUM17 (Fig. 10C).

Discussion
Corm rot is the most serious disease in saffron production, which restricts the development of saffron industry. The observation of the infection process of F. oxysporum showed that it is highly pathogenic to saffron corm under suitable growth conditions, and it may cause corm death by blocking the ducts and secreting pathogenic toxins. CAT and POD are the main protective enzymes against stress and play an important role in the antioxidant response (Boamah et al. 2021). Chitinase (CHI) and β-1,3 glucanase (β-1,3-GA) are key enzymes for plant resistance to fungal infection and are able to disrupt the cell wall of the fungus ). These resistance enzymes in saffron can play a role in the early stage of infection to resist the infection, but as the infection progresses, the resistance is difficult to maintain, which may also contribute to the susceptibility of saffron.
The cell wall is the first defensive barrier of plant against pathogens. Strengthening the cell wall through structural and chemical actions is an important resistance response of plants to fungi. The expression levels of all PAL-related Salicylic acid and jasmonic acid have been shown to play an important role in plant defense responses to F. oxysporum, and it is generally believed that salicylic acid positively regulates plant resistance . When plants are infected by fungi, salicylic acid accumulates rapidly, causing the accumulation of a series of antioxidant enzymes, such as PPO and POD, thereby protecting plants from been damaged. Wu et al. (2008) found that salicylic acid inhibited the mycelial growth, sporogenesis and conidial germination of F. oxysporum. (Dihazi et al. 2011) studied the interaction system between Trachycarpus fortunei and F. oxysporum, and found that external application of salicylic acid can cause accumulation of flavonoids and phenolic substances. In this study, the expression of salicylate receptor NPR1 was significantly increased, activating the expression of downstream PR1 protein. These results suggest that F. oxysporum infection activates the saffron salicylic acid signaling pathway to synthesize pathogenesis-related proteins and resist infection.
Most of the differentially expressed saffron transcription factors were ERF transcription factors. ERF contains a highly conserved AP2/ERF structural domain that can bind to many defense and stress response genes as well as the GCC box in the promoter region of PR genes (Singh   (Husaini et al. 2018). However, the expression levels of ERF1 and ERF2 in saffron continued to decline after infection. F. oxysporum invading hosts, first degrades the cell wall by secreting cell wall degradation enzyme (CWDE). The main cell wall degrading enzymes that have been identified are endogalacturonase, exogalacturonase, endo-xylanase and endo-pectinase. Transcriptome results showed that all these cell wall degrading enzymes genes were detected and their expression levels were significantly increased, suggesting that cell wall degrading enzymes play a key role in the infection of F. oxysporum. Toxins also promote F. oxysporum infection. They can alter the permeability of plant cell membranes, reduce the level of reactive oxygen species, prevent ATP synthesis and inhibit the growth of plants (Shao et al. 2020). When F. oxysporum was inoculated into the saffron corm, the expression level of fusarium acid synthesis genes was down-regulated. This indicates that fusarium acid is not the main toxin secreted by F. oxysporum in the early reaction of colonization compared to trichothecenes and fumonisin B1.
The interaction between plants and fungi is a dynamic and complex process. In this study, we analyzed the interaction mechanism between saffron and F. oxysporum. For F. oxysporum, it causes saffron disease by secreting cell wall-degrading enzymes, releasing multiple toxins, and activating multiple pathogenic genes. However, saffron also has corresponding response to stress. After being infected by F. oxysporum, saffron activates its own phenylpropane metabolic pathway to strengthen the cell wall, and syntheses secondary metabolites such as phenols and flavonoids, thereby inhibiting fungal growth. In addition, the plant hormone signal transduction pathway is activated, and various hormones coordinate or antagonize each other to regulate the defense against F. oxysporum. At the same time, the rapid production of reactive oxygen species, the occurrence of hypersensitivity reaction and the accumulation of various antibacterial proteins and phytoantitoxin are also important immune responses of saffron (Fig. 11). Clearly, studying the regulatory mechanism of host and pathogen interactions is beneficial to the control of saffron corm rot, and also providing a theoretical basis for mining funtional genes.
Author contributions JL, AZ, KT performed the experimental work, data curation, and formal analysis and wrote the original draft. SY, XM, and XB reviewed and revised the manuscript. YH searched the literature used in this manuscript. JB is the corresponding author and conceived the study and finalized the manuscript. All authors discussed the results and commented on the manuscript.

Funding
The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.