The potential roles of different metacaspases in maize defense response

ABSTRACT

Metacaspases (MCs), a class of cysteine-dependent proteases, act as important regulators in plant defense response. In maize genome, there are 11 ZmMCs which have been categorized into two types (type I and II) based on their structural differences. In this study, we investigated the different transcript patterns of 11 ZmMCs in maize defense response mediated by the nucleotide-binding, leucine-rich-repeat protein Rp1-D21. We further predicted that many cis-elements responsive to salicylic acid (SA), methyl jasmonate (MeJA), abscisic acid (ABA) and auxin were identified in the promoter regions of ZmMCs, and several different transcription factors were predicted to bind to their promoters. We analyzed the localization of ZmMCs with previously identified quantitative trait loci (QTLs) in maize disease resistance, and found that all other ZmMCs, except for ZmMC6-8, are co-located with at least one QTL associated with disease resistance to southern leaf blight, northern leaf blight, gray leaf spot or Fusarium ear rot. Based on previous RNA-seq analysis, different ZmMCs display different transcript levels in response to Cochliobolous heterostrophus and Fusarium verticillioides. All the results imply that the members of ZmMCs might have differential functions to different maize diseases. This study lays the basis for further investigating the roles of ZmMCs in maize disease resistance.

Programmed cell death (PCD) is a critical life process exhibiting the important role in the orchestration of cell suicide and keeping the proper function in metabolism.^1,2 The initiation of cell death program is often induced by the activation of caspases which are a family of cysteine-dependent aspartate-directed proteases functioning in the cleavage of intracellular polypeptides and the induction of stereotypic morphological and biochemical changes in cells.^3–5 However, in higher plant, there is no close homologues of caspases but caspase-like proteins, called metacaspases (MCs).⁶ MCs are conserved in plants, bacteria, fungi, and protists, but not in metazoans.² Based on the structural differences, plant MCs are categorized into two types. Type I MCs have an extension prodomain rich in proline and CxxC-type zinc-finger motifs at the N-terminus, while Type II MCs have no such structure.⁷ Both of the Type I and II MCs contain caspase-like subunits, P20 (20-kDa) and P10 (10-kDa) in the C-terminal region, and type II MCs also have a linker region between the two catalytic domains.^6,8

Many studies have demonstrated the roles of plant MCs in PCD regulation and stress responses. In Arabidopsis genome, there are nine AtMCs including AtMC 1–3 (Type I) and AtMC 5–9 (Type II).⁶ AtMC1 and AtMC2, two type I MCs, act as positive and negative PCD regulators, respectively.⁹ Overexpression of AtMC8 (type II) causes the enhancement of oxidative stress-induced PCD.¹⁰ Knockout of type II AtMCP2d/AtMC4 inhibits the PCD induced by oxidative stress and Pseudomonas syringae.¹¹ In rice (Oryza sativa) genome, eight OsMCs were identified, including OsMC1-3 (Type I) and OsMC4-8 (Type II).¹² The different OsMCs exhibit different transcript patterns under the inoculation of several pathogens or the treatment by the stress-related hormones such as abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), and ethylene (ET).¹² In addition, the tomato LeMCA1 (Type II),¹³ pepper CaMC9 (Type II),¹⁴ wheat TaMCA4 (Type II),¹⁵ and Nicotiana benthamiana NbMCA1 (Type II)¹⁶ are induced by the infection of fungal or bacterial pathogens. These studies indicate that plant MCs exert important roles in disease resistance. Recently, the maize genome is identified to contain 11 ZmMCs, including ZmMC 1–8 (Type I) and ZmMC 9–11 (Type II).¹⁷ The type I ZmMC1 and ZmMC2, but not the type II ZmMC9 suppress the autoactive hypersensitive response (HR) mediated by the nucleotide-binding, leucine-rich-repeat (NLR) protein Rp1-D21 when they were, respectively, co-expressed with Rp1-D21 in N. benthamiana.¹⁷ Rp1-D21 is derived from the intragenic recombination between two coiled-coil (CC)-NLRs, Rp1-dp2 and Rp1-D, which confers resistance to maize common rust.^18–20 Furthermore, ZmMC1 and ZmMC2 predominantly localize at the punctate structures which are partially co-localized with the autophagy maker protein ATG8, while ZmMC9 is mainly localized in the cytoplasm.¹⁷ ZmMC1 and ZmMC2, but not ZmMC9 interact with Rp1-D21 and cause the re-distribution of Rp1-D21 from nucleocytoplasm to the punctate structures in N. benthamiana and maize protoplasts.¹⁷ The results suggest that different ZmMCs might play different roles in maize defense response.

To better understand the roles of ZmMCs in Rp1-D21-mediated HR, we detected the expression patterns of 11 ZmMCs in a hybrid line containing Rp1-D21 (B73 × H95-Rp1-D21) and the corresponding wild type without Rp1-D21 (B73 × H95) via quantitative real-time PCR (qRT-PCR) analysis. We found that ZmMC5 was down-regulated and ZmMC6, ZmMC9 and ZmMC11 were up-regulated in B73 × H95-Rp1-D21 compared to wild type, while other ZmMCs had no significant change (Figure 1).

Figure 1.

The transcript levels of ZmMCs were detected by qRT-PCR using cDNA from maize seedlings of B73 × H95 WT and B73 × H95:Rp1-D21. The expression of EF1a was used as an internal control. Asterisks indicate the significant difference between samples

To further investigate the transcriptional regulation of ZmMCs genes, we analyzed the putative cis-elements in the promoter regions (2kb-upstream of the start codon) of ZmMCs by PlantCARE²¹ (plant cis-acting regulatory elements). Varieties of well-known cis-elements related to stress responses have been detected, including SA, MeJA and ABA responsiveness elements, MYB binding sites involved in drought inducibility, auxin-responsive element and gibberellin-responsive element (Figure 2 and Table 1). SA, JA and ABA are important hormones involved in abiotic and biotic stresses responses in plants.^22–25 All ZmMCs promoter regions were predicted to contain different numbers (1–8) of MeJA and ABA responsiveness elements, with ZmMC3 and ZmMC6 respectively having more ABA- and MeJA-responsive elements than other ZmMCs (Figure 2 and Table 1). ZmMC6 and ZmMC8, but not other ZmMCs were predicted to contain one SA responsive element in the promoter regions (Figure 2 and Table 1). Auxin is important for plant defense response.²⁶ In this study, auxin responsiveness element was mainly predicted in the promoter regions of ZmMC2, ZmMC8 and ZmMC9 (Figure 2 and Table 1).

Figure 2.

Cis-acting regulatory elements associated with abiotic and biotic stresses in the −2kb upstream region of the ZmMC1-11 genes were predicted by a web tool PlantCARE

Table 1.

Putative cis-elements predicted in the promoter regions of ZmMCs.

cis-elements	The number of cis-elements in the promoter regions of ZmMCs
	ZmMC1	ZmMC2	ZmMC3	ZmMC4	ZmMC5	ZmMC6	ZmMC7	ZmMC8	ZmMC9	ZmMC10	ZmMC11
MeJA-responsiveness	4	4	2	4	6	8	6	2	4	4	4
ABA-responsiveness	3	5	8	5	1	2	7	2	4	2	4
SA-responsiveness	-	-	-	-	-	1	-	1	-	-	-
Light-responsiveness	6	11	14	12	6	8	20	6	8	3	12
Low-temperature responsiveness	1	-	-	-	1	1	-	1	-	2	-
Auxin-responsiveness	1	2	-	-	-	1	1	2	2	-	-
Gibberellin-responsiveness			2	2	-	-	1	-	1	-	2
Drought-inducibility	-	1	-	-	1	2	3	1	1	-	-

We further identified many transcription factors (TFs) predicted to bind the cis-elements in the promoters of ZmMCs by JASPAR²⁰²⁰, including Dof2, Dof3, MNB1A, PBF, id1, abi4, RAMOSA1, O2 and many ARFs (Figure 3 and Table 2). Several TFs were reported to be involved in plant defense response. For example, the expressions of ZmDof were highly induced under the infection by both F. verticillioides and F. graminearum, two fungi can cause ear rot and stalk rot in maize.²⁷ abi4 belongs to APETALA2/ethylene responsive factor (AP2/ERF) class. Many TFs belong to ERF type were identified to participate in the response to biotic stresses in diverse plant species.²⁸ In addition, ARFs were also reported to function in response to biotic stress. In rice, OsARF12 and OsARF16 positively regulate the resistance to rice dwarf virus (RDV), while OsARF11 exerts negative regulation in RDV-resistance.²⁹ Infection of rice RNA viruses such as southern rice black streaked dwarf virus, rice stripe virus and rice stripe mosaic virus can be promoted by the interaction of virus proteins (P8, P2 and M) with OsARF17.³⁰ The binding sites of Dof were predicted in high frequency in the promoter regions of ZmMC1, ZmMC3, ZmMC5, ZmMC8, ZmMC10 and abi-binding sites were mainly distributed in the promoter regions of ZmMC4, ZmMC5, ZmMC6. ZmMC6 and ZmMC8 respectively contained more binding sites of ARF14 and ARF18 in their promoter regions than other ZmMCs, while ZmMC7 contained most binding sites of ARF4, ARF16 and ARF29 in the promoter region. These results implied that ZmMCs might act in maize disease resistance to different pathogens through transcriptional regulation.

Figure 3.

The different TFs predicted to bind to the promoters of the ZmMC1-11 genes as predicted by JASPAR²⁰²⁰. Different colors stand for the binding sites of various TFs

Table 2.

The binding sites of transcription factors predicted in the promoter regions of ZmMCs.

Transcription factors	The number of binding sites for transcriptionfactors
	ZmMC1	ZmMC2	ZmMC3	ZmMC4	ZmMC5	ZmMC6	ZmMC7	ZmMC8	ZmMC9	ZmMC10	ZmMC11
Dof2	4	1	5	1	4	2	2	5	2	4	-
Dof3	3	-	-	-	-	-	1	-	-	3	-
abi4	1	3	2	5	5	6	-	-	2	1	-
ARF16	1	1	-	1	2	-	3	-	-	1	1
ARF14	-	1	1	-	-	4	-	-	-	-	-
ARF18	-	-	-	-	1	-	3	4	-	-	2
ARF4	2	3	3	2	-	1	4	1	-	2	1
ARF29	-	-	2	2	-	-	4	-	1	-	-

In maize, many disease resistance associated quantitative trait loci (QTLs) were mapped and a number of disease resistance genes were cloned and functionally validated in maize.³¹ We therefore investigated whether any ZmMCs were localized in or close to the QTLs related to maize diseases resistance. ZmMC1 was found to be located in a multiple diseases resistance (MDR) locus for southern leaf blight (SLB), northern leaf blight (NLB), gray leaf spot (GLS)³² and Fusarium ear rot disease (FER)³³ on chromosome 1 (Figure 4 and Table 3). ZmMC2 was distributed in a QTL for SLB resistance³⁴ on chromosome 2 (Figure 4 and Table 3). Three closely linked Type I ZmMCs, ZmMC3, ZmMC4 and ZmMC5, were all located in GLS-resistance locus³² on chromosome 1 (Figure 4 and Table 3). ZmMC9 was in the Fusarium ear rot (FER) disease-resistance locus³³ and ZmMC10 was located in the NLB and SLB-resistance loci^34,35 (Figure 4 and Table 3). ZmMC11 was in an MDR locus for SLB, NLB, GLS and FER^32,33,36 on chromosome 4 (Figure 4 and Table 3). The transcript levels of different ZmMCs in response to different pathogens were investigated in previous RNA-seq experiments.^37,38 It was found that ZmMC10 was up-regulated while most ZmMCs except for ZmMC1 were down-regulated at 24 h after inoculated by Cochliobolous heterostrophus, which can cause SLB in maize³⁷ (Table 3). After Fusarium verticillioides infection, the transcript levels of ZmMC1, ZmMC2 and ZmMC4 were significantly induced in a maize resistant inbred line than those in the susceptible line, while ZmMC3 and ZmMC8 were down-regulated³⁸ (Table 3).

Figure 4.

The co-localization between ZmMCs and QTLs associated with disease resistance in maize genome. ZmMC1-5, ZmMC9-11 were localized close to/within the QTLs for northern leaf blight (NLB), southern leaf blight (SLB), gray leaf spot (GLS), and Fusarium ear rot (FER) disease. The lines marked by different colors stand for the regions of QTLs detected on maize chromosomes by different authors as noted in the main text

Table 3.

Maize disease-resistance QTLs in which ZmMCs located

Gene name	Gene ID	Type	Transcripts	Location	Log2 Fold change (SLB 24 h/Con 24 h)37	Log2 Fold change (Co441 Fv/Co354 Fv)38	QTLs for maize diseases
ZmMC1/ZmMCAIa	GRMZM2G155422	I	1	Chr1:95114193–95118437	0.527	3.15	Multiple diseases resistance locus for FER SLB, NLB and GLS32,33
ZmMC2/ZmMCAIb	GRMZM2G035928	I	1	Chr9:114218886–114224143	−0.555	3.18	SLB-resistance loci34
ZmMC3/ZmMCAIc	GRMZM2G120069	I	1	Chr1:67061317–67063237	−2.365	−1.22	GLS-resistance loci32
ZmMC4/ZmMCAId	GRMZM2G120079	I	2	Chr1:67083208–67085025	−1.979	2.287	GLS-resistance loci32
ZmMC5/ZmMCAIe	GRMZM2G125314	I	1	Chr1:67134761–67136668	−3.578		GLS-resistance loci32
ZmMC6/ZmMCAIf	GRMZM2G132238	I	2	Chr9:122122722–122124369	−6.256
ZmMC7/ZmMCAIg	GRMZM2G337548	I	1	Chr9:120767369–120769555	−3.393
ZmMC8/ZmMCAIh	GRMZM2G320206	I	2	Chr9:120668467–120671022	−1.923	−5.08
ZmMC9/ZmMCAIIa	GRMZM2G047274	II	1	Chr3:188387408–188390357	−1.183		FER-resistance loci33
ZmMC10/ZmMCAIIb	GRMZM2G066041	II	1	Chr8:120397497–120399854	1.274		NLB, SLB-resistance loci34,35
ZmMC11/ZmMCAIIc	GRMZM2G022799	II	1	Chr4:186662827–186664371	−2.542		Multiple diseases resistance locus for SLB, NLB, GLS and FER32,33,36

In conclusion, we have investigated the transcript patterns of 11 ZmMCs in maize defense response, the prediction of the cis-elements related to biotic stress in their promoter regions and the TFs bound to their promoters, the localization of ZmMCs with previously identified QTLs in maize disease resistance, and their transcript levels in response to different pathogens. All the results implied that the members of ZmMCs might have differential functions in response to different maize diseases. The further validation of the function of ZmMCs in maize disease defense responses will benefit for the understanding of the molecular mechanisms of MCs in plant innate immunity and provide the theoretical references for the breeding of new varieties with resistance to maize diseases.

Funding Statement

This study is supported by grants from the National Natural Science Foundation of China (31871944, 32072405 and 31571263), a Qilu Scholarship from Shandong University of China (11200086963061).

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.