Pathways related to plant immune response
So far, it has been proved that plant immune response is relative to plant-pathogen interaction, plant hormone signal transduction, and pathways about certain secondary metabolite biosynthesis or metabolism[58, 63-66]. Consistently, we got similar results in this study (Table 1).
In plant-pathogen interaction pathway, all except WRKY1/2 were up-regulated. They were CDPK (calcium-dependent protein kinase), Rboh (respiratory burst oxidase homolog), CNGC (cyclic nucleotide gated channel), calcium-binding protein CML (calmodulin-like protein), LRR (leucine-rich repeat) receptor-like serine/threonine-protein kinase FLS2, MEKK1 (mitogen-activated protein kinase kinase kinase 1), MKK4/5 (mitogen-activated protein kinase kinase 4/5), WRKY transcription factor 33, WRKY transcription factor 22, RIN4 (RPM1-interacting protein 4), serine/threonine-protein kinase PBS 1, molecular chaperone HtpG. Biological processes these up-regulated genes principally involved were hypersensitive response (HR), cell wall reinforcement, defense-related gene induction, phytoalexin accumulation and miRNA production. Some of these genes were involved in PAMP-triggered immunity. Only WRKY transcription factor 2 displayed down-regulated expression, and it was connected with HR, defense-related gene induction and programmed cell death.
In plant hormone signal transduction, we learned that GH3 (auxin responsive glycoside hydrolase 3 gene family), AHP (histidine-containing phosphotransfer protein), ARR-B (two-component response regulator ARR-B family), PIF4 (phytochrome-interacting factor 4), ERF1 (ethylene-responsive transcription factor 1), JAZ (jasmonate ZIM domain-containing protein) were up-regulated. AUX1 (auxin influx carrier), ARF (auxin response factor), CRE1 (cytokinin receptor enzyme), DELLA protein, PP2C (protein phosphatase 2C), EIN2 (ethylene-insensitive protein 2), BZR1/2 (brassinosteroid resistant 1/2), JAR1 (jasmonic acid-amino synthetase), COI1 (coronatine-insensitive protein 1), transcription factor TGA showed down-regulated. As it described, transcription factor TGA is connected with disease resistance[72]. DEGs in this pathway involved many biological processes, such as cell enlargement, plant growth, cell division, shoot initiation, stem growth, stomatal closure, seed dormancy, fruit ripening, senescence, monoterpenoid biosynthesis, indole alkaloid biosynthesis, cell elongation, of course, disease resistance as well (Fig.6). Above biological processes usually accompanied by phosphorylation (+p), dephosphorylation (-p), ubiquitination (+u). Phosphorylation and ubiquitination are common post-translational modification of proteins. They play an important role in pattern-triggered immunity (PTI), and simultaneously be necessary to receptor complex activation signals and cell homeostasis[73]. Phytohormone played a vital role in this pathway. They included jasmonic acid (JA), salicylic acid (SA), ethylene (ET), brassinosteroid (BR), auxin, cytokinine, gibberellin, abscisic acid.
In fact, plant hormones do play a vital role in the process of plant-pathogen interaction. The current study found a large number of DEGs annotated to signal transduction mechanisms by means of functional annotation. Furthermore, lots of DEGs were markedly enriched into plant hormone signal transduction pathway. Consistently, it has been reported that auxin[74, 75], cytokinins[76, 77], ethylene[75, 78-80], gibberellin[81], abscisic acid[75, 82, 83], brassinosteroids[80], salicylic acid[75, 78, 84], jasmonic acid[75, 78, 84-86], strigolactones[87] can actively participate in disease response. Among them, salicylic acid signal transduction and jasmonic acid/ethylene signal transduction are considered as the most common plant hormone signal transduction pathways in response to biological or abiotic stress. It could even be said that the plant resistance against pathogen is initially stimulated by gene expression regulated by transcription factors and ultimately be mediated by plant hormones. Therefore, if possible, it is necessary to study phytohormone metabolism of G. elata Bl. f. glauca in the following work.
Brassinosteroid is one of crucial phytohormone closely related to plant growth and stress response. In brassinosteroid biosynthesis pathway, CYP90D2 (steroid 3-oxidase) showed up-regulated expression; CYP90A1 (cytochrome P450 family 90 subfamily A polypeptide 1) displayed down-regulated expression; CYP734A1/BAS1 (PHYB activation tagged suppressor 1) was mix-regulated, with two genes up-regulated and one gene down-regulated (Fig.7).
The current study also found numerous DEGs appears in the pathways of secondary metabolites biosynthesis. CYP75B1 and CYP75A showed significant differential expression in flavone and flavonol biosynthesis pathway. 4CL, CYP84A appeared in phenylpropanoid biosynthesis pathway. 4CL and CYP73A displayed positive regulation in ubiquinone and other terpenoid-quinone biosynthesis pathway. 4CL belongs to the plant phenylpropane derivative, which is related to the synthesis of flavonoids and lignin and is a key enzyme in the biosynthetic pathway. It was reported that Fm4CL-like 1 is involved in secondary cell wall development and lignin synthesis and it play an important role in osmotic stress by affecting cell wall and stomatal development[88]. This may be a part of fungal disease immune response mechanism in G. elata Bl. f. glauca.
In starch and sucrose metabolism pathway, DEGs involved in fructose and glucose synthesis were mainly positively regulated, and they were fructokinase (EC:2.7.1.4), beta-fructofuranosidase (EC:3.2.1.26), hexokinase (EC:2.7.1.1), phosphoglucomutase (EC:5.4.2.2) and UTP-glucose-1-phosphate uridylyltransferase (EC:2.7.7.9); while several DEGs involved in starch and glycogen synthesis mainly showed negative regulation, and they covered 1,4-alpha-glucan branching enzyme (EC:2.4.1.18), starch synthase (EC:2.4.1.21), 4-alpha-glucanotransferase (EC:2.4.1.25) and so on.
In summary, fungal disease immune response is a complex process involving multiple biological processes. It covers more than one gene and one gene does not worked in single pathway. That is to say, one gene may perform more than one function simultaneously. These significantly enriched pathways would well reveal the underlying immune response mechanism of fungal disease in G. elata Bl. f. glauca.
Defense-related transcription factors
It has been proved that many a transcription factor can directly or indirectly regulate plants immune response[41-60]. Here, the current study got the similar result (Fig.4). Exceptionally, according to transcription factor prediction, some C3H genes were differentially expressed in two groups. However, present reports about C3H are mainly related to cold resistance, rather than disease resistance[61, 62].
Resistance genes (R genes)
Resistance genes (R genes) were classified into nine types based on intracellular and extracellular pathogen recognition mechanisms[89]. Here, the current study discovered potential R genes in G. elata Bl. f. glauca were probably the member of transcription factor families like WRKY, GH3, TIFY/JAZ, CML, ERF, TGA. Coincidently, it has been reported that above transcription factors did be widely involved in various defense responses[58, 90-102]. It is reported that GH3 and CML can also regulates fruit development[103, 104]. To verify the accuracy of transcriptome sequencing, qRT-PCR test was performed, and the results were basically consistent with transcriptome sequencing (Fig.10). However, it still needs further study on how these genes perform their functions in respond to fungal disease in G. elata Bl. f. glauca.
Potential immune response mechanism of fungal disease in G. elata Bl. f. glauca
Plant immune response mechanisms mainly include PAMP-triggered immunity (PTI), effector-triggered immunity (ETI) and systemic acquired resistance (SAR). ETI is usually accompanied by the occurrence of hypersensitivity reaction (HR), giving rise to programmed cell death (PCD). Moreover, ETI can induce SAR. As is known to all, PTI and SAR are non-specific immunity, while ETI is specific immunity[105]. From what the current study, the immune response mechanism of fungal disease in G. elata Bl. f. glauca involves all above three kinds of mechanisms in the whole process of infection. In this study, many genes related to stress response and disease resistance demonstrated high expression and significant difference. They were members of certain transcription factor families, like WRKY, GH3, JAZ, CML, ERF, TGA. Furthermore, these genes were closely connected with derivatives of jasmonic acid, salicylic acid, brassinosteroid, ethylene and auxin. By BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi), it is revealed that amino acid sequences of four JAZ genes in G. elata family were highly similar to certain gene sequences in Dendrobium catenatum, Phalaenopsis equestris, Apostasia shenzhenica (Fig.9). They were all belong to TIFY10 family.