As an important member in Scrophulariaceae family, the genus Paulownia contains some fast growing tree species [1
]. Up to now, Paulownia has been introduced to many countries and areas, such as Japan, Southeast Asia, Australia, and Brazil [2
]. Taking advantage of its excellent characteristics such as high-quality, even texture, strong resistance to water stress and saline alkali, Paulownia was widely utilized in building construction, furniture, musical instruments, various craft products, and even for soil conservation, windbreak, and sand fixation [3
Nevertheless, the production of Paulownia would suffer severe or even fatal loss when it is infected with phytoplasmas [5
]. Phytoplasmas are single-cell prokaryotic organisms without a cell wall, which can infect more than 1000 plant species such as jujube [6
], Paulownia [7
], mulberry [8
], peanut [9
] and sweet potato [10
]. As obligate parasites, they live and multiply not only in the intestinal tract, lymph, salivary glands and other tissues of insects, but also in the phloem cells of plants [11
]. The host plants of phytoplasmas exhibit mainly stem section shortening, leaf chlorosis, phyllody, and witches’ broom [13
]. The symptoms in phytoplasmas-infected plants might be caused by nutrient consumption, which was directly related to the activity of effector proteins secreted by phytoplasmas [15
]. Since phytoplasmas have no cell wall, they can directly interact with their host plants by exposing their membrane proteins or effectors to plant cytoplasm [16
]. Previous research indicated that phytoplasmas can regulate the gene expression levels by secreting the effector proteins TENGU, SAP11, and SAP54 to induce typical symptoms in host plants [17
]. In order to defend pathogen invasion, the corresponding immune responses were subsequently launched in host plants. So far, two immune responses against pathogen have been reported in host plants, i.e., effector-triggered immunity (ETI) and pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) [19
]. During the past few decades, some proteins, metabolites, miRNAs and genes related to plant hormone signal transduction, secondary metabolism, and photosynthesis have been found to be involved in Paulownia witches’ broom (PaWB) [20
], while the interaction between phytoplasmas and infected plants is not yet clearly known.
The gene expression network is usually regulated by multiple factors at transcriptional levels. Among all these regulatory factors, microRNAs (miRNAs, a kind of small noncoding RNAs (21–24 (nucleotides) nt) that are derived from pre-miRNA (50–350 nt)) play vital roles in gene expression regulation. Generally, miRNAs inhibit the translation process by binding to the 3′ untranslated regions of their target genes [25
]. In addition, miRNAs are also reported to influence many biological processes in biotic/abiotic stress responses [27
]. For instance, miRNA393 was found to help the host plant cells to recognize the characteristics of pathogens, and then triggered a series of defensive responses such as rapid gene expression changes, and phytohormone and metabolite induction [29
]. MiR156 and miR164 were induced in response to virus infection in Nicotiana tabacum
] and Arabidopsis thaliana Heyn
]. At the same time, miR156, miR159 and miR172 were differentially expressed in phytoplasmas infection, which played critical roles in Jujube witches’-broom (JWB) disease [6
Advances in high-throughput sequencing technologies have made the integrated analysis on small RNAs and transcriptome sequencing easier. Moreover, the availability of the Paulownia fortunei (P. fortunei) genome information has enhanced the accuracy of gene annotations in Paulownia. In this study, we combined transcriptome and small RNA (sRNA) sequencings to conduct a comprehensive analysis on the immunity mechanisms of PaWB in P. fortunei, and attempt to elucidate a miRNA-target gene interaction network related to PaWB. Overall, our results provide a platform to better understand the interaction of pathogen–host in Paulownia and other tree species.
The availability of reference genome sequences makes the annotation of important genes, proteins, lncRNAs (LncRNAs (long non-coding RNAs) are defined as having more than 200 nucleotides and little protein-coding potential, which are an important class of pervasive genes involved in a variety of biological functions [48
].), metabolites, and miRNAs more precise compared with the transcriptome background, as well as providing meaningful insights into genome reorganization, gene evolution and comparative genomic analyses between multiple species [50
]. Thus, based on the reference genome, transcriptome and miRNAs were performed in phytoplasama infected plantlets, and obtained several key genes related to PaWB. Previous studies about genes related to PaWB have been found, but the mechanisms were still unclear. To get a deeper understanding of the interaction between Paulownia and phytoplasmas, we compared the data obtained in this study with others [51
]. The number and species of miRNAs or genes in the present study were different. The probable reasons for this difference are as follows: three biological replicates were performed in this study, making the data more reliable; the plant materials in previous reports were treated with reagent, which might lead to the changes in miRNAs and gene expression; and high-throughput sequencing was performed with the genome of P. fortunei
as the reference.
4.1. PaWB Responsive DER-Target Gene Pairs Regulate Morphological Variations
Expression variations of miRNAs make it possible for responding to phytoplasmas invasion with reprogramming of gene expression. In this work, six DERs that may be related to plant defense or morphogenesis were selected to construct the miRNA-target gene interaction network based on gene function annotation. Previous studies demonstrated that downregulation of squamosa promoter binding protein-like 8 (SPL8) could increase branch development by facilitating the formation of axillary buds [53
] and regulate the GA signaling pathway and biosynthesis in stem elongation [54
]. Gou et al. [53
] indicated that GA signaling transduction was regulated by SPL8 from the upstream GA receptor (GID1) to the downstream responsive genes (GRASs). GID1 sensed and bound endogenous GA to induce the formation of GID1–GA–DELLA protein complex [53
]. There was increasing evidence that the GA-GID1 complex caused rapid degradation of the DELLA protein [55
] and DELLA as a nuclear transcription regulator could inhibit GA signaling and restrict plant growth [56
]. In addition, it was also reported that SPL regulate cell proliferation in leaf primordial [57
]. Leaf cell proliferation was positively controlled by functional transcriptional coactivators Ans and Grf-Interacting Factor 1 (GIF1) that affected the gene expression for leaf growth based on the interaction network [58
]. In this study, pf-miR2646 was upregulated in the phytoplasma-infected seedlings, which might decrease the expression level of its target gene (SPL8). Thereby, we hypothesize the downregulation of SPL8 inhibit the GA signal which may result in the stem section shortening in infected P. fortunei
. Meanwhile, regulation of leaf cell proliferation by SPL may be involved in the occurrence of witches’ broom.
Research has also shown that auxin responsive genes participated in biotic stresses to regulate the plant morphology. The involvement of ARFs in response to biotic stresses was also revealed in Arabidopsis thaliana
] and Oryza sativa
]. In addition, Fan et al. [63
] suggested that the concentration of Indole-3-acetic acid (IAA) in PaWB-infected plants was significantly lower than that in healthy plants, while the concentration of cytokine (CK) in PaWB-infected plants was significantly higher than that in healthy plants. Moreover, the upregulation of CK/IAA could lead to the appearance of witches’ broom symptoms. This is consistent with the results of that achieved in this study, i.e., ARFs (the target genes of pf-miR160h), are downregulated in the PFI library, implying that ARF TFs might play essential roles in response to PaWB-infection.
4.2. Regulation of Plant Defense Systems by DER-Target Genes Pairs
DERs modulate multiple biological processes not only in morphology, but also in the plant defense responsive to pathogen invasion. With the parallel evolution process of pathogen, the pattern of plant defense is constantly diversifying. Plants can establish a complete defense system to resist the invasion of pathogens by activating cascade response of protein kinases, strengthening the cell wall and building an salicylic acid (SA)-dependent immune response. As for strengthening the cell wall, the composition of the cell wall allows it to act as a major barrier to resist the invasion of pathogens [64
]. Zhong et al. [65
] elucidated that over-expression of MYB can induce the gene expression changes of phenylpropanoid biosynthesis, which modulate the biosynthesis of lignin, one of the components of the cell wall [66
]. In Arabidopsis, the ectopic expression of MYB TFs AtPAP1 leads to increased gene expression of pheammonia-lyase, chalcone synthase and dihydroflavonol 4-reductase, which could increase the production of ligin and anthocyanin compounds [68
]. Evidence indicated that SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1) and its homologues such as NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (Nst1), Nst2, VASCULAR RELATED NAC-DOMAIN 7(VND7) and VND6 were the main switches activating a group of MYB TFs (including MYB20, MYB46, MYB85, MYB103), which in turn aroused the secondary wall synthesis process [65
]. In this study, pf-miR858a and pf-miR858b, whose target genes encoded MYB TFs (Arabidopsis MYB58 homolog), were downregulated, and the MYB genes displayed a higher expression level in PFI than that in PF plantlets. In consideration of the above-mentioned facts, we speculate that MYB and SND1 may work together on the synthesis of the cell wall to protect against pathogen invasion in P. fortunei
As sessile organisms, plants have evolved unique cell surface receptors, which can sense chemical signals of intercellular communication to cope with various stresses. Receptor-Like Kinase (RLK) protein with a special structure (containing a signal sequence, an amino-terminal domain with a transmembrane region and a carboxyl-terminal kinase domain) is one of the dominant cell surface receptors that can receive signals of cell-to-cell communication. In Arabidopsis, leucine-rich repeat- receptor-like protein kinases (LRR)-RLKs containing an extracellular LRR and a Ser/Thr kinase domain were thought to be involved in plant defense processes via various signal cascade response [71
]. Recently, it has been reported that LRR domains of RLK could interact with multiple proteins resulting in signal response transduction [73
]. MLO proteins are leucine-rich peptides and play key roles in the signal recognition process of plant defense [73
]. The binding of the endogenous plant elicitor peptides (PEPs) to their receptors (PEPRs) located on the plasma membrane resulted in an increase of cytosolic Ca2+
, which was the early response of the signal cascade to activate the immune response [75
]. It also had been reported that the Ca2+
increase played a critical role in MLO function [77
]. Peterhansel et al. [78
] suggested that the development of MLO-mediated resistance required the involvement of two genes, ROR1 and ROR2, while MLO-mediated defense response might involve one or more small GTP-binding proteins of the ROP family [79
]. Therefore, Bhat’s [80
] research team believed that MLO, ROR2 and other proteins might develop a new pathogen-triggered microdomain at the site of biotic stress. Hence, among the interaction network when plant encounter stresses, the protein kinase family with leucine-rich repeat domain may interact with MLO to activate the signal cascade transduction to respond to phytoplasma invasion. In this study, the MLO6 (target of pf-miR164b-5p) is upregulated in PFI, indicating that the plant could trigger a series of defense responses when infected with phytoplasmas.
Salicylic acid (SA) plays an important role in protecting pathogen infection [81
]. As the key speed limiting enzyme in the biosynthesis of SA, Isochorismate synthase 1 (ICS1) was regulated by AtTCP8 [82
]. TCP8 showed positive regulation on the expression of ICS1 when interacting with the WRKY family [82
]. Moreover, in this interaction network, pf-miR2628 plays a key role in regulating the expression of WRKY4 (whose function was directly improved plant defense). In a word, during the process of PaWB-infection, pf-miR2628 and its targets may function as a key regulator to mediate the plant defense.
Taken together, our results revealed the significant roles of miRNA-target gene pairs in response to PaWB based on the interaction network, but further studies still need to be performed to explore their complex functions in Paulownia.
4.3. AS Events Related to PaWB Response in P. fortunei
AS is a vital regulatory mechanism at the post-transcriptional level in eukaryotes, which increases transcriptome complexity and protein diversity by splicing the same pre-mRNA [83
]. Research showed that AS can adjust the function of important plant stress–response components [84
]. In order to response to the invasion or attack from pathogens and pests, plant evolved defense mechanisms which involve multiple signal cascades and protein networks to provide specific and comprehensive defenses.
Serine/arginine-rich (SR) proteins belong to a conserved RNA-binding protein family, and play important roles in both constitutive splicing and AS. Recent studies have shown that alternative splicing of SR proteins play vital roles in stress response process [86
]. Xu et al. [86
] have proved that MOS14 functioned as the nuclear import receptor of SR proteins, which play vital roles in RNA metabolism. MOS14 mutation changed the splicing patterns of resistance (R) gene SNC1 and RPS4, and then damaged the plant resistance, which was mediated by these two genes. As mentioned above, SR proteins can act as splicing factors for AS [89
], and they also play a vital role in the plant immunity response.
In this study, the genes encoding SR proteins (PAU016740.2, PAU000297.1, and PAU019143.1) contained five AS categories (TSS, XSKIP, SKIP, AE, and XAE) in the PFI library. Taken together, these results suggest that AS events may influence the defense mechanisms of plants in response to pathogen invasion. We speculate that PaWB-infected plants could induce lots of SR proteins through AS events in order to respond pathogen invasion. In general, our results elucidate the crucial roles of AS events in answering to PaWB and provided new ideas to further investigate the transcriptome complexity