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2022 Volume 2
Article Contents
Abstract
Introduction
Myb transcription factors involved in the lignin biosynthesis pathway
Phylogenetic analysis of myb repressors of the lignin biosynthesis pathway in gymnosperms
Domains of myb repressors from the lignin biosynthesis pathway in gymnosperms
Myb repressors from gymnosperms are functionally different than angiosperms
Regulation of lignin myb suppressors by light quality in conifers – myb expression and lignin synthesis
Conclusions
Author contributions
Data availability
Acknowledgments
Conflict of interest
Supplementary information
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References
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ARTICLE   Open Access    

Comparative transcriptome and proteome analysis of lily clones inoculated with Fusarium oxysporum f. sp. lilii

  • 1.

    National Engineering Research Center for Ornamental Horticulture, Yunnan Flower Breeding Key Laboratory, Flower Research Institute, Yunnan Academy of Agricultural Sciences, Kunming 650200, Yunnan, China

  • 2.

    School of Agriculture, Yunnan University, Kunming 650200, Yunnan, China

  • # These authors contributed equally: Yiping Zhang, Xin Wang

More Information
  • Basal bulb rot is the major factor restricting the production of lily, caused by Fusarium oxysporum f. sp. lilii. To systematically analyze the transcriptomic and proteomic responses of lily clones to F. oxysporum, we constructed six RNA-seq libraries and four iTRAQ proteomic libraries using lily resistant and susceptible clones sampled at 0, 24 and 48 h post-inoculation (hpi) with F. oxysporum respectively. 137,715 unigenes were generated, of which 7,667 were differentially expressed. 1,679 and 4,051 differentially expressed genes (DEGs) between resistant and susceptible clones were isolated from samples collected at 0 and 48 hpi. Four hundred and thirty three and 155 DEGs were identified in resistant clones sampled at 24 and 48 hpi separately while 550 and 799 DEGs were isolated in the susceptible clones sampled at 24 and 48 hpi respectively. The results of iTRAQ analysis revealed 7,482 proteins in resistant and susceptible clones. Data analysis of transcriptome and proteome indicated that 5,735 proteins corresponded to mRNAs. Three hundred and sixteen and 1,052 DEGs had corresponding DEPs. At 48 hpi, the resistant clones showed 155 DEGs and 108 corresponding DEPs, while the susceptible clones showed 799 DEGs and 316 corresponding DEPs. In general, these results enhance comprehension of the defense response of lily resistant clones to F. oxysporum infection and provide valuable sequence data for studying the resistance mechanism.

  • Introduction

    Lignin is one of the most abundant secondary metabolites present in the cell walls of specialized plant cell types in vascular plants[1]. Lignin is an organic polyphenolic polymer that is formed by the polymerization of three monolignols – p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, that produce p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) subunits, respectively. Gymnosperms lack the S-lignin subunits and have relatively higher lignin content compared to the angiosperm woody species[2]. Lignin provides structural support, transports water and minerals, and protects the plant from pathogens, thereby acting as a barrier[3]. However, lignin forms one of the major hurdles for the forest-based industry e.g. paper, pulp, and biofuel production[4]. For example, lignin negatively affects paper quality where the presence of lignin causes discoloration of the paper and weakens it. The degradation of lignin is difficult as it is a complex polymer, therefore modification or pretreatment of lignin is required to make the wood suitable for biofuel production[5]. Hence, the detailed mechanisms involved in the regulation of lignin synthesis are valuable for the industry. Conifers form the major source of softwood for timber and paper production, and they are especially preferred for pulp because of the long fibers in their wood[6]. Even so, while the lignin biosynthetic pathway is well studied in model plants like Arabidopsis thaliana (Arabidopsis) and other angiosperm woody trees, this research area remains relatively unexplored in gymnosperms.

    The lignin biosynthesis pathway is regulated by a complex network of transcription factors from the MYB (myeloblastosis) family that either positively or negatively controls lignin synthesis[7]. In this review, MYB members were referred to that activate or suppress lignin synthesis as MYB activators or MYB suppressors/repressors, respectively. This review focuses on the potential and differential domains present in the MYB suppressors in gymnosperms along with their phylogenetic analysis. The details of all the sequences included for the domain and phylogenetic analysis are included in Supplemental Table S1.

    MYB transcription factors involved in the lignin biosynthesis pathway

    MYB family members are functionally diverse, and apart from regulating the lignin biosynthesis pathway they also control various processes involved in plant growth and development[8,9]. The N-terminal region of the MYB proteins is highly conserved containing the MYB repeats (R) involved in DNA binding; there are three types of R repeats – R1, R2, and R3. The MYB family in plants is classified into four classes according to the presence of the number of R domain repeats: 1R-MYB or MYB-related (having R1/R2/R3), R2R3-MYB (having R2 and R3), 3R-MYB (having R1, R2 and R3), and 4R-MYB (having R1, two R2 and R1/R2)[8]. The C-terminal region of the MYB proteins is highly variable and contains the regulatory domain (activation/suppression domain)[8].

    While there are several MYB members that positively regulate lignin synthesis, only a few from the R2R3-MYB class and their homologues in plant species including woody trees, negatively regulate lignin synthesis[10]. Arabidopsis is the most well studied plant model, where the R2R3-MYB members are well characterized. The R2R3-MYB class in Arabidopsis is the largest among the MYB family with 126 members which contains the basic helix-loop-helix (bHLH) domain within the R3 region. The R2R3 class is further classified into 25 subgroups depending on the motifs in the C-terminal region[9]. Subgroup 4 of the R2R3-MYB class comprises four members in Arabidopsis - MYB3, MYB4, MYB7, and MYB32[11,12]. These members contain conserved MYB motifs - LLsrGIDPxT/SHRxI/L (C1 motif) at the end of the R3 repeat and the C2 motif (pdLHLD/LLxIG/S) in the C-terminal regions. The C2 motif harbors the LxLxL-type or DLNxxP-type repression motif (Ethylene-responsive element binding factor-associated Amphiphilic Repression abbreviated as EAR)[13−15]. MYB4, MYB7, and MYB32 additionally possess a putative zinc-finger motif (ZF motif, CX1–2CX7–12CX2C) and a conserved GY/FDFLGL motif (part of the C4 motif) in their C-termini region[14]. MYB3, MYB4, MYB7, and MYB32 have been demonstrated to function as the transcriptional repressors of phenylpropanoid pathway - lignin biosynthesis pathway and/or the biosynthesis of pigments in Arabidopsis[11,12]. MYB3 negatively regulates cinnamate 4-hydroxylase (C4H) that catalyses the second step of the phenylpropanoid pathway leading to lignin and pigment synthesis, while MYB4 can inhibit almost all the enzymes in the lignin synthesis pathway causing a decrease in lignin synthesis[10,16]. R2R3-MYB members from other angiosperm species that negatively regulate lignin biosynthesis included in this review are MYB156/MYB221 from populus (Populus trichocarpa), EgMYB1 from eucalyptus (Eucalyptus gunnii), ZmMYB31/ZmMYB42 from maize (Zea mays) and PvMYB4 from switchgrass (Panicum virgatum)[10,17−20].

    Similar to the repressors, the R2R3-MYB members that act as activators of the lignin biosynthesis pathway possess the conserved R2, R3, and bHLH domains in their N-terminal region, and their C-terminal region is highly variable. But unlike the repressors, where the characteristic repressor motif e.g. EAR has been described, the presence of a specific activator motif has not been described in the R2R3-MYB activators of the lignin biosynthesis pathway in multiple angiosperm species such as Arabidopsis (MYB58, MYB63)[21], eucalyptus (EgMYB2)[22] and populus (Populus tomentosa, PtoMYB92, PtoMYB216)[23,24]. However, a few activator motifs such as SG7 and SG7-2[9,25] have been reported in the C-terminal of R2R3-MYB members belonging to subgroup 7, which positively regulates flavonoid synthesis e.g. in grapevine (VvMYBF1)[25] and Arabidopsis (AtMYB12 and AtMYB111)[9].

    Both, the repressor and activator R2R3-MYB members, repress or activate the genes from the lignin biosynthesis pathway respectively, by binding to the AC elements (adenosine and cytosine-enriched sequences) present in the promoters of lignin biosynthetic genes[26]. Another way in which these R2R3-MYB members function is by interacting with Glabrous 3 (GL3), which is the key element of the MYB-bHLH-WD40 (MBW) complex that regulates the lignin biosynthesis pathway, flavonoid biosynthesis and trichome development in Arabidopsis[27]. The repressors compete with the activators to bind to GL3 or to the AC elements of promoters, to bring about the repression of the lignin biosynthesis pathway genes[12,28].

    Apart from repressors and activators, the R2R3-MYB family comprises members that act as master regulators of cell wall formation, e.g. MYB46 which is a multifaceted R2R3-MYB transcription factor in Arabidopsis. MYB46 along with its paralogue MYB83, functions as a master switch for the secondary cell wall biosynthesis that not only mediates the transcriptional network involved in the secondary cell wall formation, but also regulates the genes from the cellulose, hemicellulose and lignin biosynthesis pathways including upstream regulators and downstream targets[29].

    Phylogenetic analysis of MYB repressors of the lignin biosynthesis pathway in gymnosperms

    To date, only two studies in gymnosperms have validated the repressor activity of the R2R3-MYB transcription factor in the lignin biosynthetic pathway - GbMYBR1 in Ginkgo biloba (Ginkgo) and CfMYB5 in Chinese cedar (Cryptomeria fortunei Hooibrenk)[30,31]. Recently, differential regulation of the MYBs (copies of MYB3 and MYB4) that potentially act as suppressors and the variation in lignin synthesis in response to light quality in the Norway spruce (Picea abies)[32] and Scots pine (Pinus sylvestris)[33] seedlings were reported based on the transcriptomic and Fourier transform infrared (FTIR) analysis. The Scots pine reads in the previous study[33] were aligned to the loblolly pine (Pinus taeda) genome (v1.01)[34], therefore the corresponding MYB sequences retrieved from loblolly pine were considered for this review. MYB3 copies from Picea were named Pa_AtMYB3-like1, Pa_AtMYB3-like2, and so on, while copies from Pinus were named Pt_AtMYB3-like1, Pt_AtMYB3-like2, and so on. A similar naming convention was followed for the MYB4 copies from both conifers. A total of 23 MYB repressors from gymnosperms including eight sequences from Norway spruce, 13 sequences from loblolly pine and one sequence from Ginkgo and one sequence from Chinese cedar were recruited for the analysis.

    The earlier phylogenetic analysis suggested GbMYBR1 to be a distinct MYB suppressor closely related to MYB5 from Arabidopsis[30] and showed that CfMYB5 was grouped with ZmMYB31 (Zea mays), EgMYB1 (Eucalyptus grandis) and AtMYB4 (Arabidopsis), which inhibited lignin synthesis[31]. For the current review, MYB members reported by earlier studies[11,12,17−20,24,30,32,33,35] were included in the phylogenetic tree (Fig. 1), which was constructed using Phylogeny.fr with default settings[36]. MYB members from Arabidopsis that repress the phenylpropanoid pathway such as MYB3, MYB4, MYB7 and MYB32 (AtMYB3, AtMYB4, MYB7, MYB32) were included in the phylogenetic tree as Arabidopsis is the most well-studied model system in plants. CfMYB5 and GbMYBR1 were included in the phylogenetic tree as they are the R2R3-MYB repressor genes from gymnosperms that negatively regulate the lignin biosynthesis pathway[30,31]. MYB5 from Arabidopsis (AtMYB5) was included in the construction of the phylogenetic tree as some of the MYB members from conifers showed the presence of the provisional MYB5 repressor in the C-terminal domain in the Conserved Domain Database[37] (CDD) search and GbMYBR1 is closely related to MYB5 from Arabidopsis[30]. The MYB3/MYB4 copies from Norway spruce and Scots pine (corresponding Pinus taeda sequences) from earlier studies[32,33], which were proposed to repress lignin synthesis, were included in the phylogenetic tree. R2R3-MYB family members from a few other species that repress lignin biosynthesis were included in the phylogenetic tree, e.g. Potri_MYB156 and Potri_MYB221 from populus; EgMYB1 from eucalyptus; PvMYB4 from switchgrass; ZmMYB31 and ZmMYB42 from maize[10,17−20]. PtoMYB170 and PtoMYB216 from Populus tomentosa and, AtMYB58 and AtMYB63 from Arabidopsis, which positively regulates lignin deposition during the formation of wood[21,35], were included in the phylogenetic tree as an outgroup.

    Figure 1.  Phylogenetic tree constructed with copies of MYB3-like and MYB4-like from Picea abies (Pa) and Pinus taeda (Pt) along with GbMYBR1 from Ginkgo biloba (Gb); CfMYB5 from Cryptomeria fortune (Cf); MYB3, MYB4, MYB5, MYB7, MYB32, MYB58 and MYB63 from Arabidopsis thaliana (At); MYB156 and MYB221 from Populus trichocarpa (Potri); MYB170 and MYB216 from Populus tomentosa (Pto); EgMYB1 from Eucalyptus gunnii (Eg); ZmMYB31 and ZmMYB42 from Zea mays (Zm) and, PvMYB4 from Panicum virgatum (Pv).
    DownLoad: Full-Size Img PowerPoint

    The phylogenetic tree (Fig. 1) shows two distinct sub-clades, one sub-clade that contains all the MYB3-like members from the two conifers (except Pt_AtMYB3-like7) and one MYB4-like member from spruce (Pa_AtMYB4-like3), along with AtMYB5 and GbMYBR1. The other sub-clade includes all the MYB4-like members from the two conifers along with AtMYB3 and AtMYB4 from Arabidopsis, and the R2R3-MYB family suppressors from populus, eucalyptus, switchgrass, and maize. Overall, the phylogenetic analysis shows a clear separation of the MYB3-like and MYB4-like R2R3-MYB members from the two conifer species into two groups, where the GbMYBR1 from Ginkgo groups with the MYB3-like members. This suggests that the MYB3-like members from conifers and Ginkgo may have distinct motifs which differ from the motifs present in the angiosperms. However, the MYB4-like conifer members seem to have motifs that are similar to angiosperm species e.g. many of the MYB4-like conifer members contain the EAR motif, while EAR was detected in only one of the MYB3-like members (Pt_AtMYB3-like1). CfMYB5 from Chinese cedar seems to contain unique motifs compared to all the gymnosperm members included in this study.

    Domains of MYB repressors from the lignin biosynthesis pathway in gymnosperms

    Alignments of MYB repressors from the gymnosperms and angiosperms species are included in the Supplemental information (Supplemental Figs S1–S5). GbMYBR1 shows distinct sequence characteristics; it has low identity with characterized MYB4 repressors from Arabidopsis and other angiosperm species. Although the characteristic domains of the R2R3-type repressors e.g. C1, C2, ZF, and C4 motifs and, the typical repressors motifs like the LxLxL-type EAR motif and the TLLLFR motif are absent in GbMYBR1 from the C-terminal, GbMYBR1 has the R2 and R3 domain in the N-terminal region including the conserved bHLH-binding motif (Fig. 2)[30]. CfMYB5 from Chinese cedar contains the conserved R2 and R3 domains in the N-terminal like Ginkgo, however, the study did not report any typical R2R3-type suppressor domain in its C-terminal[31]. The current sequence analysis reports the presence of the EAR suppression domain (LCLSL) in the C-terminal region of CfMYB5 (Fig. 3, Supplemental Fig. S5), which is novel.

    Copies or homologs of MYB3 and MYB4 were detected to be differentially regulated under shade (Low Red : Far-red) in Norway spruce and Scots pine and these MYB copies were proposed to repress the lignin synthesis as their down-regulation correlated with enhanced lignin synthesis[32,33]. Alignments[38] performed with the different copies of MYB repressors reported by earlier studies[8,30,32,33] show that the sequences are well conserved in gymnosperms and angiosperms in the N-terminal regions (R2, R3 along with the bHLH binding motif) but not in the C-terminal region (Fig. 2, Supplemental Figs S1−S5) which are in accordance with the previous findings. The C-terminal regions (with the C1, C2, ZF, and C4 motifs) are the most variable regions within the different conifer MYB members (Supplemental Figs S1−S4) which agrees with the findings in Arabidopsis[8,9]. The alignment of partial C-terminal regions of MYB repressors from gymnosperms and angiosperms (Fig. 3) show that most MYB3/MYB4 copies from both conifers lack the classical LxLxL-type EAR motif. It is worth noting that generally monocots possess the LNLDL motif and dicots have the LNLEL motif, while the conifers show the presence of both LNLDL and LNLEL, in addition to four more patterns – LNLNL, LDLGL, LDLQL, and LQLLL (Fig. 3). In Pt_AtMYB4-like1, Pt_AtMYB4-like3, Pt_AtMYB4-like4, and Pa_AtMYB4-like1, either LNLNL/LNLDL/LDLGL, LNLNL/LNLEL or LNLDL/LDLQL or LNLNL/LNLEL could function as a potential repressor, respectively (alternative EAR domains are marked with a box and, bold and underlined for Pt_AtMYB4-like1, Pt_AtMYB4-like3, Pt_AtMYB4-like4, and Pa_ AtMYB4-like1 in Fig. 3). It is also possible that LNLNLDLGL and LNLNLEL could function as a unique type of lignin repressor in conifers. CfMYB5 from Chinese cedar also shows the presence of a new pattern of the EAR motif (LCLSL), which was not described previously in this species by earlier investigations and is not represented in the angiosperms. None of the gymnosperm MYB members contain the conserved TLLLFR motif or the GY/FDFLGL motif, which are essential for the repressor activity of the transcription factors (Supplemental Figs S4 & S5)[12,14]. This is similar to the unique kind of R2R3-MYB-type repressor reported recently in Ginkgo (GbMYBR1)[30]. Similar to GbMYBR1, all the MYB3/MYB4 copies in both the conifer species contain the bHLH binding motif in the R3 region that could potentially be involved in the repression mechanism[30].

    Figure 2.  Alignment of N-terminal regions of the lignin repressor MYB members from gymnosperms and angiosperms showing the conserved R2, R3, and bHLH domains: MYB3-like and MYB4-like copies from Picea abies (Pa) and Pinus taeda (Pt) along with GbMYBR1 from Ginkgo biloba (Gb); CfMYB5 from Cryptomeria fortune (Cf); MYB3, MYB4, MYB7, and MYB32 from Arabidopsis thaliana (At); MYB156 and MYB221 from Populus trichocarpa (Potri); EgMYB1 from Eucalyptus gunnii (Eg); ZmMYB31 and ZmMYB42 from Zea mays (Zm) and PvMYB4 from Panicum virgatum (Pv).
    DownLoad: Full-Size Img PowerPoint
    Figure 3.  Alignment of partial C-terminal regions of the lignin repressor MYB members from gymnosperms and angiosperms showing the conserved EAR domain (alternative EAR domains in Pt_AtMYB4-like1, Pt_AtMYB4-like3, Pt_AtMYB4-like4, and Pa_AtMYB4-like1 are marked with box and, bold and underlined): MYB3-like and MYB4-like copies from Picea abies (Pa) and Pinus taeda (Pt) along with GbMYBR1 from Ginkgo biloba (Gb); CfMYB5 from Cryptomeria fortune (Cf); MYB3, MYB4, MYB7, and MYB32 from Arabidopsis thaliana (At); MYB156 and MYB221 from Populus trichocarpa (Potri); EgMYB1 from Eucalyptus gunnii (Eg); ZmMYB31 and ZmMYB42 from Zea mays (Zm) and PvMYB4 from Panicum virgatum (Pv).
    DownLoad: Full-Size Img PowerPoint

    Except for two MYB members in Pinus (Pt_AtMYB3-like7 and Pt_AtMYB4-like3), all the other MYB sequences in both conifers possess either the EAR repressor motif in the C-terminal (Fig. 3) and/or the putative repressor domain PLN03212 in the N-terminal region (Supplemental Table S1). The PLN03212 domain was detected in the motif search using CDD[37] with a very low E-value. The PLN03212 domain is the provisional repressor domain for the transcription factor MYB5 in Arabidopsis, but this domain has not been functionally characterized. MYB5 represses the flavonoid pathway, regulates mucilage synthesis, and plays a role in the development of the seed coat and trichome morphogenesis[39]. The PLN03212 domain is also present in GbMYBR1, but its potential function in the process of repression has not been reported[30]. The PLN03091 was yet another domain that was detected in the searches with CDD with a very low E-value in MYB suppressors from both conifers, similar to lignin repressors of other plant species (Supplemental Table S1). The PLN03091 is denoted as a provisional hypothetical protein in the CDD.

    It is proposed that conifers contain different types of MYB3/MYB4-like repressors, some of which contain the classical repressor motifs and others without the repressor motifs (e.g. LxLxL) analogous to that of GbMYBR1. Similar to GbMYBR1, the MYB3/MYB4-like repressors detected in Norway spruce and Scots pine[32,33] might have distinct sequence characteristics or motifs, whose potential functional characterisation needs further validation. These repressors and their mode of regulation especially regarding the defense and phenylpropanoid pathways might be unique to conifer species, which also needs further characterization.

    MYB repressors from gymnosperms are functionally different than angiosperms

    The repressors from the MYB family are not fully explored in gymnosperms unlike the well-studied model plants e.g. Arabidopsis. GbMYBR1 from Ginkgo is mainly expressed in young leaves, although its expression was detected in the roots, stem and fruits; the expression level of GbMYBR1 in young leaves were more than 12-fold higher than the levels in roots or stems. GbMYBR1 is a unique kind of R2R3-MYB-type repressor that lacks the characteristic repressor motifs e.g. EAR motif and the TLLLFR motif from the C-terminal region, yet it suppresses the lignin biosynthesis pathway. Overexpression of GbMYBR1 in Arabidopsis represses lignin synthesis specifically through down-regulation of the key lignin biosynthesis pathway gene – hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) which encodes the enzyme that catalyzes the rate-limiting step of the lignin biosynthesis pathway. In addition, GbMYBR1 down-regulates a few other genes from the lignin biosynthesis pathway including phenylalanine ammonia-lyase (PAL), 4-coumarate: CoA ligase (4CL) and cinnamyl alcohol dehydrogenase (CAD). GbMYBR1 overexpression also reduces the pathogen resistance by significantly down-regulating a great number of defence-related genes in the transgenic Arabidopsis. Moreover, the transgenic Arabidopsis overexpressing GbMYBR1 was more susceptible to bacterial infection as compared to the wild type. However, the regulatory process of the GbMYBR1 is entirely different from Arabidopsis; the lignin synthesis suppression by GbMYBR1 is not only more specific compared to the MYB repressors in Arabidopsis but the mode of action of GbMYBR1 to repress lignin synthesis is different from Arabidopsis[30]. The GbMYBR1 mode of action is mediated through direct and specific interaction with GL3 to compete against the interaction of GL3 with MYB activators, leading to the suppression of lignin synthesis[30]. For example, in Arabidopsis, the MYB4 that represses the lignin biosynthesis pathway, physically interacts not only with GL3 but also with other bHLH cofactors (e.g. TT8 and EGL3) to bring about the suppression[40]. Thus, the interaction of Arabidopsis repressor MYB with bHLH cofactors is not as specific as for GbMYBR1. In addition, overexpression of GbMYBR1 led to the down-regulation of HCT – a key gene from the lignin biosynthesis pathway along with only a few other genes from the lignin biosynthesis pathway in contrast to other angiosperm species where expression of multiple genes was affected along with reduced lignification as a result of the overexpression of MYB that acts as a repressor[30]. Thus, the suppression of GbMYBR1 on lignin biosynthesis is more specific than for the other repressor MYBs in Arabidopsis. Su et al. proposed the working model of GbMYBR1 and presented the details on the regulatory mechanism of GbMYBR1 in transgenic Arabidopsis[30], yet whether GbMYBR1 directly regulates the genes from lignin biosynthetic pathway in Ginkgo needs to be further explored and validated.

    CfMYB5 from Chinese cedar which negatively regulates the lignin biosynthesis is a nucleus-localized protein that is expressed at higher levels in the stem as compared to the needle, bud, male cone, and root[31]. The repressor activity of CfMYB5 was demonstrated from the analysis of its expression patterns; overexpression of CfMYB5 in the transgenic lines correlated with the decrease in expression of the key genes involved in the lignin biosynthesis pathway (HCT, PAL, 4CL, and CAD) along with a decrease in secondary cell wall formation which involves the deposition of both lignin and cellulose. Thus, CfMYB5 suppression was not specific only for lignin synthesis.

    Regulation of lignin MYB suppressors by light quality in conifers – MYB expression and lignin synthesis

    MYB4 has been demonstrated to respond to light quality in Arabidopsis; for example, UV-B irradiation down-regulates MYB4[13]. As the MYB repressor is involved in the negative regulation of the lignin biosynthesis pathway, its down-regulation leads to higher lignin synthesis. In Norway spruce, down-regulation of two copies of MYB3 under shade (Low Red : Far-red ratio) in the northern populations correlated with higher lignin synthesis in the case of north vs south comparisons[32] (Supplemental Table S1). While none of the repressors were detected to be differentially regulated under shade in the southern population, an equal number of repressors (MYB3/MYB4) were found to be up-regulated and down-regulated (p-value > 0.05) in the northern population of Norway spruce[32]. Nevertheless, MYB3/MYB4 gene expression in Scots pine was not fully in favour of higher lignin synthesis under shade[33]. The Scots pine reads in a previous study[33] were aligned to the loblolly pine (Pinus taeda) genome (v1.01)[34], therefore the corresponding MYB sequences retrieved from loblolly pine were considered for this review, as mentioned previously. Therefore, it is important to note that the Pinus taeda information in Supplemental Table S1 corresponds to Scots pine. An equal numbers of repressors (MYB3/MYB4) were found to be up-regulated and down-regulated (p-value > 0.05) respectively in the southern and northern Scots pine population under shade (Supplemental Table S1). In the case of north vs south comparisons, four MYB members were found to be up-regulated in the northern Scots pine population as compared to the southern population under shade, suggesting higher lignin synthesis in the southern population[33]. The analysis for genes that positively regulate the lignin biosynthesis in these studies suggest an equal number of genes being up-regulated and down-regulated under shade in both the conifer species[32,33]. However, the FTIR spectroscopic data confirmed higher synthesis of lignin in response to shade as compared to the sun conditions in both conifers[32,33]. The difference in the binding capacity between the MYB family members may be one of the possible reasons behind the inconsistency between MYB3/MYB4 expression and lignin synthesis. A proteomic and metabolomic analysis may reveal concordance between the FTIR data and the MYB3/MYB4 regulatory mechanism. In addition, it is the highly variable C-terminal region of different plant MYBs that contains the repressor domain, which is not characterized in conifers. For example, in Arabidopsis, a change (D261N) in a conserved amino acid in the GY/FDFLGL motif present in the C-terminal region of the R2R3-type MYB4 transcription repressor resulted in abolishing its repressive activity[14]. The N-terminal region of the different MYB members in both conifers was found to be conserved while the C-terminal region was highly variable (Supplemental Figs S1−S5). It is proposed that conifers may contain novel motifs in the C-terminal region of the MYB members that may be specific to conifer species, which needs to be functionally validated[41]. Furthermore, there could be conifer-specific co-repressors that interact in general with the MYB members of subgroup 4 and specifically with MYB3 to regulate the phenylpropanoid pathway similar to Arabidopsis where the NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED1 (LNK1) and LNK2 act as co-repressors along with MYB3[42]. The LNK-MYB3 transcription complex plays a role in the repression of the C4H gene, one of the key genes involved in lignin biosynthesis[42]. Other factors contributing to the phenylpropanoid pathway regulation includes interactions between the MYB members that are co-expressed, their probable interactions with other transcription factors and the feedback loops. These need further investigation in conifers. Similar arguments were proposed for the detection of several grass MYB4 homologs binding to the promoters of genes involved in the lignin biosynthesis pathway, which was not in accordance with the expression of the MYB4 genes[43,44].

    The increase in lignin synthesis in response to shade in conifers[32,33] is a contrasting feature compared to angiosperms, where shade causes a decrease in lignin synthesis due to which the angiosperm becomes weak and susceptible to diseases[45,46]. The underlying mechanism, whether and how the MYB repressors may be involved in this process in conifers needs further research.

    Conclusions

    The sequence analysis suggests that although the domains of the MYB repressors from the lignin biosynthesis pathway are conserved among the angiosperms and gymnosperms in their N-terminal regions, they may possess diverse repressor domains in the C-terminal regions that have not been functionally characterized. Gymnosperms are ancient and functionally diverse compared to angiosperms in many ways. For example, comparative genome annotation studies revealed notable differences in the size of the NDH-complex gene family and the genes underlying the functional basis of response to shade suggesting specialization of the photosynthetic apparatus in Pinaceae[47]. Likewise, it is proposed that the lignin biosynthesis pathway in conifers may function through alternative mechanisms, unlike those observed in the angiosperms, as suggested by the study in Ginkgo[30]. Further investigation is required for functional validation of all the conifer MYB repressors discussed in this review aiming to elucidate the mechanisms underlying the repression of the conifer lignin biosynthesis pathway.

    Author contributions

    The authors confirm contribution to the paper as follows: study conception and design: Ranade SS; García-Gil MR; data collection: Ranade SS; analysis and interpretation of results: Ranade SS; draft manuscript preparation: Ranade SS; García-Gil MR. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    All data generated or analyzed during this study are included in this published article and its supplementary information files.

    Acknowledgments

    We acknowledge the support from FORMAS (FA-2021/0038) and Knut and Alice Wallenberg Foundation.

  • The authors declare that they have no conflict of interest.

  • Supplemental Table S1 Primers used for Q-PCR.
    Supplemental Fig. S1 Gene Ontology (GO) enrichment analysis of differentially expressed genes. A, GO enrichment analysis in profile SLC-00 vs RLC-00; B, GO enrichment analysis in profile SLC-48 vs RLC-48; C, GO enrichment analysis in profile SLC-00 vs SLC-24; D, GO enrichment analysis in profile SLC-00 vs SLC-48; E, GO enrichment analysis in profile RLC-00 vs RLC-24; F, GO enrichment analysis in profile SLC-00 vs RLC-48.
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  • Cite this article

    Zhang Y, Wang X, Jin C, Xu F, Yang X, et al. 2022. Comparative transcriptome and proteome analysis of lily clones inoculated with Fusarium oxysporum f. sp. lilii. Ornamental Plant Research 2:23 doi: 10.48130/OPR-2022-0023
    Zhang Y, Wang X, Jin C, Xu F, Yang X, et al. 2022. Comparative transcriptome and proteome analysis of lily clones inoculated with Fusarium oxysporum f. sp. lilii. Ornamental Plant Research 2:23 doi: 10.48130/OPR-2022-0023
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Comparative transcriptome and proteome analysis of lily clones inoculated with Fusarium oxysporum f. sp. lilii

  • 1.

    National Engineering Research Center for Ornamental Horticulture, Yunnan Flower Breeding Key Laboratory, Flower Research Institute, Yunnan Academy of Agricultural Sciences, Kunming 650200, Yunnan, China

  • 2.

    School of Agriculture, Yunnan University, Kunming 650200, Yunnan, China

  • Received: 22 August 2022
  • Accepted: 07 November 2022
  • Published online: 28 December 2022
Ornamental Plant Research  2,  Article number: 23  (2022)  |  Cite this article

Abstract: Basal bulb rot is the major factor restricting the production of lily, caused by Fusarium oxysporum f. sp. lilii. To systematically analyze the transcriptomic and proteomic responses of lily clones to F. oxysporum, we constructed six RNA-seq libraries and four iTRAQ proteomic libraries using lily resistant and susceptible clones sampled at 0, 24 and 48 h post-inoculation (hpi) with F. oxysporum respectively. 137,715 unigenes were generated, of which 7,667 were differentially expressed. 1,679 and 4,051 differentially expressed genes (DEGs) between resistant and susceptible clones were isolated from samples collected at 0 and 48 hpi. Four hundred and thirty three and 155 DEGs were identified in resistant clones sampled at 24 and 48 hpi separately while 550 and 799 DEGs were isolated in the susceptible clones sampled at 24 and 48 hpi respectively. The results of iTRAQ analysis revealed 7,482 proteins in resistant and susceptible clones. Data analysis of transcriptome and proteome indicated that 5,735 proteins corresponded to mRNAs. Three hundred and sixteen and 1,052 DEGs had corresponding DEPs. At 48 hpi, the resistant clones showed 155 DEGs and 108 corresponding DEPs, while the susceptible clones showed 799 DEGs and 316 corresponding DEPs. In general, these results enhance comprehension of the defense response of lily resistant clones to F. oxysporum infection and provide valuable sequence data for studying the resistance mechanism.

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    INTRODUCTION

    • Lily (Lilium spp.) is a floricultural crop with great ornamental value, playing a vital role in global flower markets. Lily basal bulb rot, caused by the soil-borne fungal pathogen Fusarium oxysporum f. sp. Lilii, is one of the most severe diseases of lily plants worldwide[1]. Chemical pesticide control is one of the main methods to prevent and control this disease whereas the cultivation of Fusarium resistant varieties is more economical and effective as shown by numerous field practices. The resistant cultivar, such as oriental lily hybrid 'Casa Blanca', has been achieved by screening lily clones with the toxin of F. oxysporum[2]. However, the mechanism underlying F. oxysporum f. sp. lilii resistance of the resistant clones remains unknown.

      Plants recognize F. oxysporum infection by detecting endogenous signals originating from the cell wall[3,4]. Disease-resistant enzymes, cell wall proteins, pathogenesis-related (PR) proteins and phytoalexin biosynthetic enzymes play a key role in pathogen defense in host plants[5−8]. Pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) released from the microbial surface can bind to pattern recognition receptors (PRRs) present in plant cells and activate them[9,10]. Plants also employ chemical defense pathways to resist pathogen infection. For example, melon plants induce genes encoding chitinases (CHIs), β-1,3-glucanases, thaumatin-like proteins and peroxidases (PODs) upon the infection of F. oxysporum[4]. Phytohormones such as salicylic acid (SA) and jasmonic acid (JA), as well as other antioxidants are also induced during the pathogen infection as a defense mechanism[6,11]. In addition to the above factors, sugar was also reported to be involved in the plant disease signaling pathways[12,13].

      To date, only limited studies have been published about the F. oxysporum f. sp. Lilii resistance mechanism. A transcriptome study on Lilium regale Wilson with the infection of F. oxysporum revealed that genes encoding PR proteins, signal transduction proteins, antioxidative stress enzymes and secondary metabolism enzymes were involved in the F. oxysporum response[14]. Further study of L. regale identified a novel basic leucine-zipper (bZIP) transcription factor LrbZIP1, which could be important for F. oxysporum resistance in L. regale as the transgenic tobacco plants overexpressing LrbZIP1 showed increased resistance to F. oxysporum[15]. Besides, PR10 family genes related to defense responses to F. oxysporum f. sp. lilii were also identified in L. regale[16].

      Transcriptional analysis has been applied to investigate the mechanism underlying plant disease-resistance[17−19]. The iTRAQ technology enables high-throughput analysis of proteins with high sensitivity, providing highly reliable results[20]. Thus, the successful application of RNA-seq and iTRAQ technologies offers a great opportunity to isolate genes responsible for F. oxysporum resistance in lily[21].

      In this study, we sequenced six RNA-seq libraries prepared from F. oxysporum resistant and susceptible lily clones sampled at 0, 24 and 48 h post-inoculation (hpi) with F. oxysporum by the Illumina sequencing platform, and analyzed four proteome libraries of these clones sampled at 0 and 48 hpi using ITRAQ technology. Additionally, qRT-PCR was used to verify the RNA-seq data and to evaluate the transcriptional variation between the lily resistant and susceptible clones. The results of this study reinforce the understanding of lily molecular mechanism resistance to F. oxysporum.

    MATERIALS AND METHODS

      Plant materials

    • Lily resistant and susceptible clones were obtained by screening oriental lily hybrid 'Casa Blanca' with the toxin of F. oxysporum. The resistant and susceptible clones were grown in the same culture chamber at 25 °C with the 14 h light/10 h dark photoperiod and robust tissue culture rooted seedlings were selected for further analysis; rooted seedlings are 8 cm high, with a root length of 5 cm.

      Cultivation and inoculation of Fusarium oxysporum f. sp. lilii

    • Fusarium oxysporum f. sp. lilii was isolated from the infected lily. The isolated pathogen was purified and identified according to the spore and mycelium morphology. The verified strain was then cultured iteratively using potato sucrose liquid medium every 15 d on a shaker (25 °C, 100 rpm). The cultured spores were filtered by gauze and resuspended, then diluted to a final concentration at approximately 1.0 × 106 spores mL−1 by microscopic count. The root tips of in vitro lily clones were cut off 2 cm and inoculated by the spore suspension. The inoculated clones were then transferred to climate chambers at 25 °C with the 14 h light/10 h dark photoperiod.

      Sample collection

    • Based on our previous results[1], plant samples were collected from regions surrounding the inoculation spots at 0, 24 and 48 hpi for transcriptomic analysis and at 0 and 48 hpi for proteomic analysis. Three independent biological replicates were assayed at 24 and 48 hpi. Total RNA was extracted with Trizol and was treated with DNase. The quality of total RNA was identified by 2100 Bioanalyzer. Library construction and sequencing was carried out in accordance with Hao et al.[22]

      RNA-seq data analysis and de novo assembly

    • Raw sequence reads were removed to obtain clean reads. Then, de novo transcriptome assembly was carried out[23]. RNA-Seq by RSEM software was used to analyze the gene expression level[24,25].

      Functional annotation of unigenes

    • The identified unigene sequences were aligned and annotated using the following databases (E-value ≤ 10−5): NCBI nonredundant protein sequences, NCBI nucleotide sequences, Swiss-Prot[26], Gene Ontology[27], Kyoto Encyclopedia of Genes and Genomes[28], Cluster of Orthologous Groups of proteins and euKaryotic Ortholog Groups[29], Protein family (Pfam).

      Analysis of differential expression of Unigenes

    • The Unigene expression level of the samples were compared, and the generalized chi-square test was performed using IDEG6 software, and the P value obtained was corrected by multiple hypothesis test (FDR). Then, the Unigenes whose FDR value is less than 0.01 and the RPKM ratio between samples is more than 2 times as the differentially expressed gene.

      Identification of RNA-seq data by qRT-PCR

    • To identify the reliability of RNA-seq data, the expression of nine genes, including five genes upregulated in lily resistant clones and four PR genes (encoding CHI [gi691193462], POD [gi636022329], polyphenol oxidase [PPO; gi767859558] and PAL [gi393793951]) was analyzed by qRT-PCR. The Actin gene was used as an internal control gene. Primers were designed by Primer Premier 5 software (Supplemental Table S1). The qRT-PCR was made by SYBR Premix Ex Taq II according to the following program: initial denaturation at 95 °C for 2 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. Relative mRNA levels were calculated by the 2−ΔΔCᴛ method[30].

      Protein extraction and iTRAQ labeling

    • Protein extraction was conducted by the trichloroacetic acid (TCA)/acetone method. Protein concentration was determined by the Bradford method[31]. Then, 200 μg of protein solution was accurately absorbed by pipette into the centrifuge tube. Add 4 μl reducing reagent, reaction at 60 °C for 1 h; Add 2 μl cysteine-blocking reagent, room temperature for 10 min; it was then transfered to a 10-K ultrafiltration tube and centrifuged at 12,000 g for 20 min. The 100 μl dissolution buffer in iTRAQ® kit was added and centrifuged at 12,000 g for 20 min. Trypsin was added into the ultrafiltration tube, the volume of 50 μl, 37 °C reaction overnight; The next day, the solution at the bottom of the tube was collected after centrifugation. A total of 100 μl samples were obtained after enzymolysis.

      iTRAQ® reagent was taken from the refrigerator, balanced to room temperature, centrifuge the iTRAQ® reagent to the tube bottom; Add 150 μl ethanol to each iTRAQ® reagent, centrifuge it to the bottom of each tube. The 50 μl sample (100 μg enzymolysis product) was transferred to a new centrifuge tube with a pipette. The iTRAQ® reagent was added to the sample, vortexed, and centrifuge to the bottom of the tube, at room temperature for 2 h. The reaction was terminated by adding 100 μl water. The mixed labeled samples were centrifuged to the bottom of the tube; Vacuum freezing and centrifugal drying; The drained samples are preserved under frozen conditions for later use.

      High pH reverse-phased chromatography

    • High pH separation was performed according to the method of Zhang et al.[32]

      Protein analysis by Eksigent Nano LC upgraded REVERSE phase chromatography -TripleTOFTM 5600

    • The peptides were resuspended and protein analysis was carried out according to the method by Tatusov et al.[31]

      Proteomics data analysis

    • ProteinPilot4.5 software (AB SCIEX, Foster City, California, USA) was used for protein identification and quantitative analysis. The database was downloaded from the NCBI website. Paragon algorithm was used for database retrieval. Proteinpilot software was used to carry out FDR analysis, and protein ratios and P values are calculated automatically by Proteinpilot software.

      Bioinformatics analysis

    • Enrichment analysis of the GO functional classification of differentially expressed proteins (DEPs) was mainly conducted according to the enrichment analysis of the GO functional classification of the transcriptome. Statistical analysis of the GO primary functions of all proteins with a GO number was performed. KEGG pathway enrichment analysis of the DEPs was conducted according to the results of the Lilium transcriptome.

    RESULTS

      RNA-seq read assembly

    • RNA-seq analysis of 14 samples including resistant lily clone (RLC) and susceptible lily clone (SLC) collected at 0 hpi, referred to as RLC-00 and SLC-00, RLC and SLC samples collected at 24 and 48 hpi (three biological repeats for every set), referred to as RLC-24, RLC-48, SLC-24 and SLC-48, yielded 573,692,261 raw reads, 284,383,013 reads from RLC profiles and 289,309,248 reads from SLC profiles, contributing to 563,793,160 clean reads after filtration in total. GC content of all the profiles was approximately 50%. The percentage of reads conformed to Q20 was over 90% for all profiles whereas the clean rates of the reads were no less than 94%, indicating the RNA-seq libraries had good qualities suitable for further analyses (Table 1).

      Table 1.  Statistics of RNA-seq results of inoculated lily clones.

      RNA-Seq sampleRaw- readsRead1-Q20Read2-Q20Read1-GCRead2-GCClean readsClean-rates
      RLC-0034,134,7510.9120.9700.4970.5073246945895.12%
      RLC24-159,345,1270.9650.9730.5630.5525922768299.80%
      RLC24-240,575,0870.9260.9540.5190.52340574457100.00%
      RLC24-335,961,5650.9720.9680.4710.4903571813999.32%
      RLC48-141,690,5240.9530.9580.4910.5024150937599.57%
      RLC48-236,600,1450.9740.9650.5000.5083484924895.22%
      RLC48-336,075,8140.9090.9620.5340.5403420397694.81%
      SLC-0034,363,6280.9080.9620.4740.4973260967394.90%
      SLC24-141,369,6020.9520.9570.4750.4984120339699.60%
      SLC24-278,356,0000.9270.9520.4920.50578354289100.00%
      SLC24-331,787,4830.9050.9630.4910.5033014082494.82%
      SLC48-137,014,1730.9430.9560.4790.4993691933499.74%
      SLC48-233,129,0820.9720.9760.4750.4863297114499.52%
      SLC48-333,289,2800.9700.9700.4650.4843304216599.26%

      Identification of DEGs among different profiles of RLC and SLC

    • To further investigate putative genes associated with F. oxysporum response, the DEGs between different profiles were analyzed. 1,679 genes were differentially expressed between RLC-00 and SLC-00 profiles, with 962 upregulated and 717 downregulated. There were 433 genes differentially expressed (260 upregulated and 173 downregulated) between RLC-00 and RLC-24 profiles whereas the number of DEGs was 550 (465 upregulated and 85 downregulated) between SLC-00 and SLC-24. The largest number of DEGs, 4,051 (2,035 upregulated and 2,016 downregulated) was presented between profiles RLC-48 and SLC-48 while the numbers of DEGs were 799 (558 upregulated and 211 downregulated) between profiles SLC-00 and SLC-48. Interestingly, the DEGs between profiles RLC-00 and RLC-48 were limited to 155 (89 upregulated and 66 downregulated) (Fig. 1).

      Figure 1. 

      DEGs identified between different resistant and susceptible lily clones.

      GO and KEGG enrichment analysis of DEGs

    • The huge variations in the number of DEGs between different samples implied diverse defense responses upon F. oxysporum infection, leading us to a further investigation of the putative biological functions of the identified DEGs. Those DEGs could be summarized into three major functional categories by GO annotation, meaning 'biological process', 'molecular function' and 'cellular component'. The top three subcategories in profile RLC-00 vs RLC-24 were 'DNA binding', 'monooxygenase activity' belonging to 'molecular function', and 'cell wall' belonging to 'cellular component'. Similarly, 'DNA binding' and 'cell wall' ranked in the top two subcategories in profile RLC-00 vs RLC-48, and the third subcategory was 'sequence-specific DNA binding'. Differing from RLC samples, the top three subcategories in the profile SLC-00 vs SLC-24 were 'chloroplast', 'oxidoreductase activity' and translation whereas 'structure constituent of ribosome', 'translation' and 'ribosome' were the top three in profile SLC-00 vs SLC-48. The top three subcategories in profile RLC-48 vs SLC-48 were 'cytoplasmic membrane-bounded vesicle', 'oxidoreductase activity' and 'carbohydrate metabolic process' (Supplemental Fig. S1).

      Pathways involved in plant disease-resistant were revealed by KEGG pathway analysis, including 'plant hormone signal transduction', 'cutin, suberine and wax biosynthesis', 'phenylpropanoid biosynthesis' and 'phenylalanine metabolism'. Interestingly, the 'plant-pathogen interaction' pathway was ranked first both in the profile RLC-00 vs RLC-24 and profile RLC-00 vs RLC-48 whereas this pathway was absent in SLC samples (Fig. 2a& b). The expression patterns of genes involved in this pathway were illustrated in FPKM heat map (Fig. 2c). Most of the genes were upregulated in the RLC-24 samples but remained unchanged in SLC samples, indicating that genes involved in this pathway could contribute to the resistant capacity of RLC. DEGs of KEGG was showed in RLC-00 vs RLC-48, SLC-00 vs SLC-48, RLC-00 vs SLC-00, RLC-48 vs SLC-48 (Table 2). Cysteine and methionine metabolism, protein processing in endoplasmic reticulum, ubiquitin mediated proteolysis, alpha-Linolenic acid metabolism, phenylalanine metabolism were key process in ET, SA, JA signal transduction, there were 16, 26, 8, 6, 5 DEGs respectively in the RLC-48 vs SLC-48.

      Figure 2. 

      DEGs isolated from RLC00-RLC24 and RLC00-RLC48 samples were enriched. (a) Gene number were enriched in the profile RLC-00 vs RLC-24; (b) Gene number were enriched in the profile RLC-00 vs RLC-48; (c) DEGs isolated from samples were enriched.

      Table 2.  DEGs of KEGG in different groups.

      KEGGDEGs of group
      SLC-00 vs SLC-48RLC-00 vs RLC-48RLC-48 vs SLC-48RLC-00 vs SLC-00
      Cysteine and methionine metabolism9166
      Glycine, serine and threonine metabolism31166
      Arginine biosynthesis2115
      Alanine, aspartate and glutamate metabolism71145
      Peroxisome81156
      Tyrosine metabolism114
      Phenylalanine metabolism1154
      Phenylalanine, tyrosine and tryptophan biosynthesis1142
      Valine, leucine and isoleucine biosynthesis232
      PPAR signaling pathway552
      Tryptophan metabolism333
      Arginine and proline metabolism362
      Apoptosis211
      AMPK signaling pathway5171
      Plant hormone signal transduction21
      Lysine biosynthesis221
      Valine, leucine and isoleucine degradation2181
      Plant-pathogen interaction91103
      MAPK signaling pathway - yeast442
      Endocytosis121151
      Calcium signaling pathway33
      Pathogenic Escherichia coli infection31
      Cutin, suberine and wax biosynthesis12
      Oocyte meiosis816
      TGF-beta signaling pathway212
      Endocrine and other factor-regulated calcium reabsorption65
      cGMP-PKG signaling pathway32
      Ubiquitin mediated proteolysis718
      Protein processing in endoplasmic reticulum193269
      alpha-Linolenic acid metabolism8163

      Verification of RNA-seq data

    • To verify the RNA-seq data, the expression of nine genes possibly involved in the plant defense system, including NADH dehydrogenase (ubiquinone) activity gene, ATP synthesis coupled electron transport gene, chitin-binding gene, mitochondrial respiratory chain complex I gene, response to salicylic acid gene, PPO, POD, PAL and CHI were detected by qRT-PCR. The qRT-PCR results were consistent with the RNA-seq data (Fig. 3), thus verifying that our RNA-seq data were credible. DN139810, DN144416 and PAL were expressed higher in the RLC than SLC. Notably, the expression of most of the tested genes was upregulated by F. oxysporum inoculation in RLC after 24 h rather than in SLC, implying that those genes were possibly associated with the resistance of RLC.

      Figure 3. 

      Quantitative real-time PCR (qRT-PCR) analysis of the relative expression levels.

      Protein function annotation and DEP analysis

    • Quantitative proteome analysis identified 7,482 proteins in total, 5,735 (76.65%) and 3,888 (51.96%) of which were annotated using the GO and KEGG databases, respectively. GO terms such as 'metabolic process' (3,963), 'cellular process' (3,531), 'single-organism process' (2,896), 'response to stimulus' (1,061) and 'immune system process' (112) were enriched in the biological process category; 'cell part' (3,505) and 'cell' (3,505) were enriched in the cellular component category; 'catalytic activity' (3,135) and 'binding' (2,675) were enriched in the molecular function category (Fig. 4).

      Figure 4. 

      GO classification of differentially expressed proteins (DEPs). (a) Cellular component. (b) Molecular function. (c) Biological process.

      A total of 2,055 DEPs with fold-change (FC) ≥ 2.0 and p-value ≤ 0.05 were screened. 316 DEPs (166 upregulated and 150 downregulated) were identified between RLC and SLC while 1,052 DEPs (579 upregulated and 473 downregulated) were verified between RLC-48 and SLC-48 (Fig. 5a). KEGG pathway enrichment analysis revealed DEPs enriched in different pathways. Pathways with p-values < 0.05 and < 0.01 were defined as significantly enriched pathways and very significantly enriched pathways, respectively. One hundred and fifty seven DEPs were significantly enriched in six pathways between SLC00 and RLC00 samples, including 'metabolic pathways', 'glyoxylate and dicarboxylate metabolism', 'porphyrin and chlorophyll metabolism', 'pentose phosphate pathway' and 'phenylpropanoid biosynthesis' (Fig. 5b ). Five hundred and fourteen DEPs were significantly enriched in seven pathways between SLC48 and RLC48 samples, including 'metabolic pathways', 'glyoxylate and dicarboxylate metabolism', 'carbon fixation in photosynthetic organisms', 'glycine, serine and threonine metabolism' and 'alanine, aspartate and glutamate metabolism' (Fig. 5c).

      Figure 5. 

      DEPs identified between different lily samples. (a) numbers of DEPs. (b) Numbers of DEPs. between SLC00 and RLC00 samples. (c) Numbers of DEPs. between SLC48 and RLC48 samples.

    DISCUSSION

    • Oriental lily is a highly heterozygous flowering plant with a genome size of approximately 36 Gb[21]. To fully explore the mechanism of disease resistance in Fusarium resistant lily mutant clones, we carried out comparative studies of transcriptome and proteome simultaneously to achieve complementarity and integration, which can better analyze the disease-resistance mechanism. In this study, 137,715 unigenes were identified and annotated. GO enrichment analysis indicated that these genes are involved in signal transduction mechanism, energy production and conversion, inorganic ion transport mechanism and defense mechanisms. Simultaneously, a protein search library was constructed based on transcriptome data. KEGG pathway enrichment analysis shows that DEGs and DEPs are involved in plant hormone signal transduction, oxidative phosphorylation, phenylalanine metabolism, ribosome pathway, glycine, serine and threonine metabolism, alanine, aspartate and glutamate metabolism and other resistance-related metabolic pathways. These disease resistance processes involve all aspects of plant activities, indicating that lily–F. oxysporum interaction is regulated by a multigene network[33,34].

      Different mechanisms are underlying the plant-pathogen interaction conferring the plant resistance upon infection by various pathogens. The primary difference between the resistant and susceptible hosts upon pathogen invading is their performance at the key time points, also the responding speed and defense efficiency[35]. The defense response of a susceptible host is slow and weak, which allows the pathogen to colonize the plant rapidly and induce disease[8,36]. In this study, both resistant and susceptible clones were inoculated with F. oxysporum, which induced DEGs and DEPs involved in disease resistance-related metabolic pathways. Therefore, our research focuses on metabolic pathways associated with disease resistance and the DEGs and DEPs involved in these pathways.

      Phytohormone signaling pathways

    • Plant hormones are active substances produced by plant cells in response to certain environmental factors. These include auxin, gibberellic acid (GA), cytokinin (CK), abscisic acid (ABA), ethylene (ET), ethephon (ETH), salicylic acid (SA), jasmonic acid (JA), brassinosteroid (BR) and polyamines. Plant hormones function at low concentrations by acting alone or in a synergistic manner to regulate various physiological processes such as cell division, cell elongation, tissue and organ differentiation, seed dormancy and germination, flowering, fruit production, maturation, senescence and in vitro culture[37]. Plant hormones such as SA, JA and ET, are involved in defense response against various pathogens[38,39]. Studies indicate that SA, JA and ET form a highly ordered regulatory network to regulate abiotic stress response. SA dominantly responds to biological stress by inducing the host plant system to acquire disease resistance[40], whereas JA and ET respond to biological stress by triggering induced systemic resistance (ISR)[41,42]. Many DEGs and DEPs verified in this study were involved in signal transduction and plant hormone (JA and ET) metabolic pathways. Our RNA-seq and iTRAQ data indicated that DEGs and DEPs on response to SA and SA metabolic process were upregulated, suggesting that the response to SA in lily resistant clones might be much stronger than that in lily susceptible clones.

      Plant–pathogen interactions

    • During the long-term exposure to pathogens, plants employ a series of defense mechanisms such as HR, change in defense-related enzyme levels (such as PAL and POD), production of phytoalexins (PAs) and accumulation of PR proteins[43−45]. PR proteins are induced not only after pathogen infection but also in answer to different abiotic stresses, such as drought, wounding and freezing. Among these, CHI (PR3, PR4, PR8 and PR11), either alone or in combination with beta-1,3-glucanase (PR2), can effectively enhance disease resistance in plants[46]. CHI is a typical PR protein that plays a very important role in preventing fungal pathogens from invading host plants. In general, CHI expression level in host plants is very low, but when host plants are invaded by pathogenic fungi, the level of CHI protein increases in plant cells. To date, CHI has been detected in approximately 100 plant species, and the CHI gene of many host plants has been cloned. To enhance disease resistance, foreign CHI genes have been introduced into many crop plants, such as wheat, rice and tobacco[47,48]. In this study, we found that several genes upregulated in response to F. oxysporum infection were related to the chitin catabolic process, chitin binding and CHI activity pathway. This suggests that PR genes function cooperatively to induce resistance against F. oxysporum in resistant lily clones.

      A large number of studies show that POD enzymes play an important role in biotic and abiotic stress responses[49−51]. In this study, the DEGs and DEPs on POD metabolic process were induced in lily resistant clones upon inoculation with F. oxysporum. This implies that lily resistant clones may combat the attack by F. oxysporum. Phenylpropanoid compounds, such as lignin, flavonoids and anthocyanins, play a key role in plant disease-resistance. The data showed that genes and proteins upregulated after inoculation with F. oxysporum were involved in the phenylpropanoid pathway. POD not only acts as a key antioxidant enzyme but also regulated the biosynthesis of G-, S- and H-lignin monomers[39,52]. In this study, genes encoding POD enzymes were upregulated in response to F. oxysporum inoculation.

      Role of the cell wall in disease resistance

    • Plant cells are encompassed by a thick cell wall, which provides mechanical support to cells, maintains cell morphology and is involved in various physiological activities such as extracellular signal recognition. The cell wall is the first physical barrier of plant cells against invading pathogens[33,53,54]. When host plants are infected by a pathogen, the cell wall damage signal is activated, which further activates the defense response. This leads to the production of phenolic substances and accumulation of callose and lignin in cells surrounding the infection site, thus strengthening the thickness of the cell wall to resist pathogen invasion[55]. In this study, inoculation of lily resistant clones with F. oxysporum spore suspension upregulated the expression of many genes and proteins involved in enhancing cell wall thickness.

      Regulation mechanism of resistance to Fusarium oxysporum

    • Based on our work, we put forword a hypothetical model to illuminate the resistance of lily resistant clones to F. oxysporum. In this model, the plants recognize PAMPs through their innate recognition receptors, resulting in a series of cellular responses. The plant RPM1 protein recognizes HSP90 to activates ETI to induce plant hypersensitive response (HR), at the same time, Ca2+ activates CNGCs to induce plant hypersensitive response (HR), which result in cell death and hinders further infection. The expression of cutin, suberine, wax, phenylpropanoid biosynthesis and plant hormone signal transduction pathway-related genes is activated. The expression of SA-induced disease resistance-related genes TGA increased. Glycine, serine, threonine chitin metabolism increased significantly. In summary, we supposed that the PPAR, MAPK-plant signalling pathway, SA resistance pathway, SOD metabolism pathway are involved in the defence of lily resistant clones against F. oxysporum(Fig. 6).

      Figure 6. 

      Hypothetical model of the mechanism of lily resistant clone tolerance to Fusarium oxysporum.

      Overall, in this study, we obtained information on the transcriptomic and proteomic responses of lily to F. oxysporum infection. Our results indicated that a large number of DEGs and DEPs conferring resistance to F. oxysporum were refered to SA and phenylpropanoid metabolic pathways. The DEGs and DEPs related to for defense responses also involved antioxidant enzymes, POD, PPO and PR proteins pertaining to various families. These results suggest the molecular mechanism potential of the defense response of lily against F. oxysporum and provide potential candidate gene targets for cultivar improvement by genetic engineering. Additionally, our results provide new and key sequence data for analyzing gene functions and further analyzing disease resistance mechanisms in lily.

      Acknowledgments

      • This work was supported by National Key R&D Program of China (Grant No. 2019YFD1000400, 2020YFD10004), Kunming Comprehensive test station of national Flower Industry system (CARS-23-G56), National Natural Science Foundation of China (No. 31260484), Yunnan Provincial Science and Technology Department (Grant No. 2016BB009), the Green Food Brand-Build a Special Project (Floriculture) supported by Yunnan Provincial Finance Department (530000210000000013742).

      Conflict of interest

      • The authors declare that they have no conflict of interest.

      • # These authors contributed equally: Yiping Zhang, Xin Wang

      Supplementary information
      • Supplemental Table S1 Primers used for Q-PCR.
      • Supplemental Fig. S1 Gene Ontology (GO) enrichment analysis of differentially expressed genes. A, GO enrichment analysis in profile SLC-00 vs RLC-00; B, GO enrichment analysis in profile SLC-48 vs RLC-48; C, GO enrichment analysis in profile SLC-00 vs SLC-24; D, GO enrichment analysis in profile SLC-00 vs SLC-48; E, GO enrichment analysis in profile RLC-00 vs RLC-24; F, GO enrichment analysis in profile SLC-00 vs RLC-48.
      Rights and permissions
      • Copyright: © 2022 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (6)  Table (2) References (55)
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    Cite this article
    Zhang Y, Wang X, Jin C, Xu F, Yang X, et al. 2022. Comparative transcriptome and proteome analysis of lily clones inoculated with Fusarium oxysporum f. sp. lilii. Ornamental Plant Research 2:23 doi: 10.48130/OPR-2022-0023
    Zhang Y, Wang X, Jin C, Xu F, Yang X, et al. 2022. Comparative transcriptome and proteome analysis of lily clones inoculated with Fusarium oxysporum f. sp. lilii. Ornamental Plant Research 2:23 doi: 10.48130/OPR-2022-0023
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  • Html

  • Introduction
  • Materials and methods

  • Plant materials

  • Cultivation and inoculation of fusarium oxysporum f. sp. lilii

  • Sample collection

  • Rna-seq data analysis and de novo assembly

  • Functional annotation of unigenes

  • Analysis of differential expression of unigenes

  • Identification of rna-seq data by qrt-pcr

  • Protein extraction and itraq labeling

  • High ph reverse-phased chromatography

  • Protein analysis by eksigent nano lc upgraded reverse phase chromatography -tripletoftm 5600

  • Proteomics data analysis

  • Bioinformatics analysis

  • Results

  • Rna-seq read assembly

  • Identification of degs among different profiles of rlc and slc

  • Go and kegg enrichment analysis of degs

  • Verification of rna-seq data

  • Protein function annotation and dep analysis

  • Discussion

  • Phytohormone signaling pathways

  • Plant–pathogen interactions

  • Role of the cell wall in disease resistance

  • Regulation mechanism of resistance to fusarium oxysporum

  • Acknowledgments
  • Conflict of interest
  • Supplementary information
  • Rights and permissions
  • About this article
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