Upon assaulted by pathogens, plants respond by activation of intricate defense systems. Depending on the nature of pathogens, biotrophic and necrotrophic infections are fundamentally different in terms of their infection approach, effector proteins, and the host defense response . Thus, tackling the disease based on infection stage, hypothetically should be able to save or at least prolong the life span of Ganoderma-infected palms. Identifying the infection at biotrophic phase may help planters to take suitable disease management strategy to prevent the disease from transition to the more chronic necrotrophic phase. Meanwhile, infected palms at necrotrophic phase may be treated with more intensified practice such as using chemical fungicide. Differentiation of biotrophic and necrotrophic TFs were based on established defense-related biomarkers in known defense mechanisms such as plant innate immunity and HR that can distinguish these two phases. In this study, we demonstrated the key roles of EgJUB1 as a biotrophic-specific while EgERF113 as a necrotrophic-specific transcriptional regulator, respectively during early oil palm-G. boninense interaction.
EgJUB1 mediates defense response during biotrophic phase through SNBE motif
Expression profile of EgJUB1 observed in RNA-seq was validated via qPCR which confirms its role in host defense regulation during biotrophic phase of G. boninense infection independent of abiotic stress. JUB1 was first linked to homeostasis of oxidative stress particularly related to hydrogen peroxide (H2O2) signaling. It binds to cis-element that serves as the NACBS in the promoter region of DREB2A TF for tolerance to abiotic stresses . DREB2A TF binds directly to DRE sequence of drought-stress responsive genes, including HSFs but the mechanisms are still unclear . AtJUB1 (ANAC042) was also reported as a key TF that induces camalexin expression, a major phytoalexin of Arabidopsis against bacterial pathogen . A more recent study revealed induced expression of NAC042_5, an orthologue of AtJUB1 in response to biotrophic fungus Erysiphe necator . The JUB1 which acts independent of SA was induced specifically during pathogen colonization. Intriguingly, oil palm EgJUB1 was found to co-express with two SA-dependent genes of EgTGA1 and EgNPR1. Our results were more in line with the SA-dependent master regulator of NPR1, a cofactor of TGA1 reported by , which induces Pathogenesis related (PR) genes  during biotrophic phase.
We are reporting for the first time induced expression of EgJUB1 under pathogen challenge, regulating defense-related gene(s) harbouring SNBE binding motif. SNBE motif is composed of an imperfect palindromic 19-bp sequence which can be present in various targets’ promoters including TFs and downstream genes involved in secondary cell wall biosynthesis, cell wall modification as well as PCD . Binding to the 19 bp SNBE (A/T)NN(C/T)(C/G/T)TNNNNNNNA(A/C)GN(A/C/T)(A/T) consensus sequence is critical at the 9 core nucleotides, regardless of mutation on the other nucleotides . They reported that mutation(s) on these 9 core nucleotides causes reduced and/or elimination of the transcriptional activation, on the contrary changes in the other non-critical nucleotides enhance the binding affinity.
It was discovered from our study that EgJUB1 directly regulates EgHSFC-2b to promote resistance against biotrophic phase through SNBE1 motif. The SNBE1 motif tested in this study consists of the nucleotides G and T at the 5th and 18th position of the core motif, respectively. Based on the report by Zhong et al. , the binding affinity can still be maintained if a single nucleotide in the core motif of SNBE consensus sequence is changed, however, changes of two and more nucleotides may reduce the binding affinity significantly. Thus, promoters of the listed oil palm TFs (Fig. 3D), EgHSFB-4b, EgGAMYB-X2, EgERF003, EgKAN1-like-X3, EgILI-5-like, EgERF086-like, EgPIF3-X1 and -X2, which harbour single nucleotide change at either the 5th or the 18th position of the SNBE1 core motif most likely are still able to bind to SNBE1.
Our findings are in line with a recent study which observed high up-regulation of HSF and heat shock proteins (HSPs) against biotrophic fungus [58,59]. HSF was also reported during bacterial infection which directly regulated Enhanced Disease Susceptibility 1 (EDS1) and PR4 under SA-mediated signaling .
Proposed defense-related pathways regulated by EgJUB1 co-expressing genes
Here, we report that high expression of EgCESA4, EgCESA9 and EgCSLD2 correlates with the expression of EgGAMYB-X2 TF during biotrophic phase (3 and 7 d.p.i) before a subsequent decline in expression. GAMYB TF interacts with GAMYB binding motif to activate downstream genes . The GAMYB motif was found in the promoter region of CESA responsible in secondary cell wall cellulose biosynthesis [35,62]. Consistently, CESA4, CESA7 and CESA9 were reported as regulators of secondary cell wall cellulose synthesis . In addition, local cell wall reinforcement by CSLD2 has been proven under biotroph challenge of powdery mildew fungus (Douchkov et al., 2016). Thus, it is strongly postulated that EgJUB1 binding to SNBE1 motifs in the promoter regions of EgGAMYB-X2 activates oil palm defense response through regulation of secondary cell wall biosynthesis.
Increased production of ROS accompanied with PCD , provide evidence on the occurrence of HR. In the current study, genes regulating antioxidant enzymes EgSOD, EgGSTU17, EgGST3 and EgGSTF12 were highly upregulated during early interaction with G. boninense. We also observed the expression of EgNPR1 and EgTGA1 which have been recognized to be overexpressed exclusively during biotrophic attack under SA-mediated signaling pathway [65,66]. The co-actions of EgNPR1 and EgTGA1 results in upregulation of EgPR1 which has been proven as a biotrophic marker in our previous report .
EgERF113 mediates defense response during necrotrophic phase through GCC-box and DRE/CRT motifs
MYC2 relies exclusively on co-actions of JA-ABA branch response to regulate resistance against insects and wounding by repressing the JA-ET branch . Although EgMYC2 was preliminarily screened as a necrotrophic biomarker , the TF is categorized as a downregulated DEG in our RNA-seq data. This can be explained by elevated expression of ethylene insensitive 3 (EgEIN3) which then activates EgERF113 through ET regulation. Binding of ET to its receptors inactivates CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1) which in turn release repression on EIN2 activity and stabilize EIN3 and EIN3-LIKE1 (EIL1) within the nucleus . As a result, ERFs are activated and the ERF TFs modulate transcriptional activity of developmental as well as stress-induced responsive genes . Master regulator of ethylene signaling pathways, EIN3 and EIN3-like (EIL) have been proven to modulate multitude cascades of downstream transcriptional responses [47,69]. De-repression of EIN3/EIL from JA-ZIM domain (JAZ) activates JA-ET signaling that positively regulate transcriptional activations of development as well as defense response against necrotrophic pathogens [47,70]. The findings demonstrated that oil palm establishes resistance against early necrotrophic through co-actions of JA-ET, rather than JA-ABA.
Here, we report regulation of defense response against necrotrophic phase of G. boninense through multi-cascades activation of JA-ET branch leading to overexpression of EgERF113. ERF113, also recognized as RELATED TO APETALA2.6-LIKE (RAP2.6L) which is closely related to ERF108 (RAP2.6) was found to be responsive to abiotic stresses (salinity, heat and drought) as well as stress hormones, particularly JA and/or ET . ERF108 has been reported in JA-induced defense response against wounding and pathogens [72,73]. Similarly, ERF113 was shown to be induced upon wounding [74,75], as well as pathogens infection [76,77].
We are the first reporting on EgERFF113 with binding preferences for GCC-box and DRE/CRT motifs. AP2/ERFs are known to have multiple conserved DNA binding preferences . However, DREBs are typically known to recognize DRE/CRT conferring resistance against abiotic stresses whilst ERFs bind to GCC-box promoting defense against biotic stresses. ERF113 was reported to bind to the GCC-box , but not tested on DRE/CRT motif. To date, only a few ERFs were reported with binding preferences on both GCC-box and DRE/CRT conferring resistance against pathogens attack [79,80,81,82]. Binding to GCC-box and DRE/CRT motifs that are present in plant defensin (e.g PDF1.2) as well as PR genes, activates defense-related genes [83,84]. Kaur et al. , also reported DRE/CRT element in the promoter region of calcium-responsive genes. Calcium ion (Ca2+) signaling plays paramount importance in defense mechanisms of plant in perceiving invading pathogens .
Phukan et al.  has studied the divergent of AP2/ERF TF DNA-binding specificities based on sequence characterization. Their findings suggested glutamic acid (E) at the 20th and alanine (A) at the 48th positions as identified in EgERF113 in the 60 amino acids long DBD denote binding specificity to DRE/CRT motif. EgERF113 also showed conservation of amino acids for GCC-box binding at the 10th, 18th, 20th, 37th and 59th positions. Although the specific amino acid in DBD that regulates binding of TFs to both GCC-box and DRE/CRT motifs has not yet been confirmed, replacing phenylalanine (F) at the 14th position to tryptophan (W) in EgERF113 changes the binding specificity from GCC-box only to binding both GCC-box and DRE/CRT motifs, as suggested by Phukan et al. . An additional amino acid (basic polar) at the 24th position of DBD which is lacking in EgERF113 is essential for specific binding to GCC-box only. Thus, EgERF113 with 60 amino acids DBD opposes the classification of specific GCC-box binding DBD of 61 amino acids in length.
Proposed defense-related pathways regulated by EgERF113 co-expressing genes
Two RBOH genes, EgRBOHA and EgRBOHB have been previously reported in our study , and reduced EgRBOHB expression during early necrotrophic phase may suggest plant’s response in delaying the progression of disease. This is in line with the report on the response against hemibiotroph Macrophomina phaseolina , wherein increase expression of RBOH leads to increase susceptibility of plants to necrotrophic infection.
Another less studied TF, AtMYB122 was reported to regulate genes involved in camalexin biosynthesis . Increased accumulation of indolic glucosinolates during induction of glucose signaling was evidently regulated by AtMYB122 . Glucosinates [89,90]. Based on the expression patterns of EgMYB122, the TF channeled its regulation from responding to abiotic stress at 3 d.p.i. into defensing against Ganoderma attack and it is expected that the gene regulation will be more extensive at later stage of the necrotrophic phase.
Rapid and transient increase of cytosolic Ca2+ particularly during pathogens interaction results in activation of both PTI and ETI signaling . CMLs and CDPKs are known as Ca2+ sensor proteins which are responsible in perceiving the transduction during plant innate immunity [91,92]. Based on our RNA-seq data analysis, both EgCML7 and EgCDPK28 were upregulated during necrotrophic phase. Consistent with the analysis, EgERF113 is suggested to orchestrate defense mechanisms through regulation of PR and calcium-responsive genes.
Other transcription factors differentially regulated under biotrophic or necrotrophic phase
The Calmodulin-binding transcription activator (CAMTA) gene was first discovered in Nicotiana tabacum, regulating senescence and cell death , and was recently comprehensively studied by Kakar et al. . We discovered members of the novel CAMTA TF family, namely EgCAMTA3 and EgCAMTA4 which were downregulated at 3 d.p.i. The suppression of both genes was later reduced across time, which might be the result of infection phase transition from biotrophic to early necrotrophic phase. CAMTA was proven to regulate responses under Ca2+ signaling during both abiotic and biotic stresses [95,96].
In general, most downregulated DEGs of TFs showed de-repression across time. It is reasonable to postulate that changes of expression patterns might be the results of plant immunity interplay against biotrophic and necrotrophic infection phases. For instance, downregulation of EgMYB108 was highest at 7 d.p.i before reducing at 11 d.p.i. Coherently, MYB108 was proven to positively regulate defense mechanism against hemibiotroph Verticillium dahliae in the presence of calmodulin and Ca2, antagonistic to the regulation of CAMTA3 [97,98]. In contrast, EgERF9 was found to be downregulated exclusively during necrotrophic phase at 11 d.p.i. The result provides agreement with other study which reported repression activity of AtERF9 in enhanced resistance against necrotrophic Botrytis cinerea . Likewise, expression patterns of TFs in upregulated DEGs were higher at early interaction against G. boninense before decreasing over time. The plant-specific EgTCP15 demonstrated upregulation during biotrophic phase before declining but showing an opposite expression pattern under abiotic stress.