Maize FERONIA‐like receptor genes are involved in the response of multiple disease resistance in maize

Abstract Receptor‐like kinases (RLKs) are key modulators of diverse cellular processes such as development and sensing the extracellular environment. FERONIA, a member of the CrRLK1L subfamily, acts as a pleiotropic regulator of plant immune responses, but little is known about how maize FERONIA‐like receptors (FLRs) function in responding to the major foliar diseases of maize such as northern corn leaf blight (NLB), northern corn leaf spot (NLS), anthracnose stalk rot (ASR), and southern corn leaf blight (SLB). Here, we identified three ZmFLR homologous proteins that showed cell membrane localization. Transient expression in Nicotiana benthamiana proved that ZmFLRs were capable of inducing cell death. To investigate the role of ZmFLRs in maize, we used virus‐induced gene silencing to knock down expression of ZmFLR1/2 and ZmFLR3 resulting in reduced reactive oxygen species production induced by flg22 and chitin. The resistance of maize to NLB, NLS, ASR, and SLB was also reduced in the ZmFLRs knockdown maize plants. These results indicate that ZmFLRs are positively involved in broad‐spectrum disease resistance in maize.

SLB is caused by the necrotrophic fungus Bipolaris maydis. Both NLB and ASR rank among the most devastating maize fungal diseases in the United States and Canada, causing yield losses of more than 40% in conducive environmental conditions (Mueller et al., 2016).
Furthermore, due to changes in cultivation strategies, climate, and the extensive use of susceptible maize hybrids, NLB and ASR have the potential to cause serious yield losses in maize production in countries such as China and Brazil, the second and third largest producers of maize in the world, respectively (FAO, 2019). In addition, B. zeicola and B. maydis are the major pathogens affecting maize production in China (Dai et al., 2018;Liu et al., 2015). NLS and SLB can cause yield losses of 10%-20% in years with severe epidemics (Dai et al., 2016;Sun et al., 2020).
Higher plants possess a two-layer immune system to sense varieties of immunogenic signals when infected with fungal pathogen (Boller & He, 2009). Cell-surface pattern recognition receptors (PRRs) typically perceive pathogen-/damage-associated molecules or apoplastic pathogen-associated effectors (Boutrot & Zipfel, 2017;Couto & Zipfel, 2016;Yu et al., 2017). Intracellular receptors, most commonly nucleotide-biding leucine-rich repeat proteins (NLRs), sense pathogen effectors that are delivered into the plant cell (Wu et al., 2017). Prior research generally confirms that a variety of RLKs, such as leucine-rich repeat RLKs, cell wall-associated RLKs, lectin RLKs, proline-rich extension-like RLKs, and Catharanthus roseus RLK1-like kinases (CrRLK1Ls), regulate many cellular processes during vegetative and reproductive development (Dievart & Clark, 2004;Escobar-Restrepo et al., 2007;Jose et al., 2020;Ringli, 2010). CrRLK1 was first isolated from a suspension of cells of Catharanthus roseus, and is a receptor-like protein kinase (Schulze-Muth et al., 1996). CrRLK1Ls are involved in many processes, such as cellular growth and morphogenesis, reproduction, immunity, hormone signalling, and abiotic stress tolerance (Franck et al., 2018). In Arabidopsis, all 17 members of the CrRLK1L subfamily possess an extracellular domain (ECD) with two malectin-like domains (MLD), a transmembrane domain, and an intracellular serine/threonine kinase domain (Lindner et al., 2012). The gene encoding FERONIA (FER), a well-characterized member of the CrRLK1Ls subfamily, was first cloned during the screening of double-fertilization regulators participating in pollen tube reception through reactive oxygen species (ROS) and Ca 2+ signalling (Escobar-Restrepo et al., 2007). FER is also involved in cell growth. A FER loss-of-function mutant showed obvious root hair defects (Duan et al., 2010), severe hypocotyl inhibition (Deslauriers & Larsen, 2010), and severe cell elongation defects (Guo et al., 2009). In rice, two homologous FERONIA-like receptors (FLRs) were shown to control plant morphology, fertility, and seed yield (Li et al., 2016). Furthermore, FER participates in a variety of plant hormone responses. FER employs the small G protein signalling network mediated by GEF1/4/10-ROP11 to directly activate the phosphatase activity of the key regulator ABI2 in the abscisic acid (ABA) signalling pathway, thereby negatively regulating the ABA response (Yu et al., 2012). In contrast, auxin is positively regulated by FER through the GRE-ROP/ARAC module (Duan et al., 2010).
FER also works as a prominent component in the plant immune response. Arabidopsis plants display enhanced resistance to the fungal pathogens Fusarium oxysporum and Golovinomyces (syn. Erysiphe) orontii in the absence of FER (Kessler et al., 2010;Masachis et al., 2016). In parallel, FLR2 and FLR11 mutations lead to increased resistance to Magnaporthe oryzae without growth penalty in rice plants . However, the Arabidopsis fer mutant was more susceptible to Hyaloperonospora arabidopsidis and Colletotrichum higgansianum (Kessler et al., 2010). Prior research has thoroughly investigated the role of FER in modulating the receptor kinase complex assembly, and its influence on pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), being required for the ROS burst triggered by flg22 and chitin (Stegmann et al., 2017). FER promotes the association of FLS2-BAK1 complexes and EFR-BAK1 complexes in response to flg22 and elf18, respectively (Stegmann et al., 2017). ANXUR1 (ANX1) and ANXUR2 (ANX2), which have extremely high sequence similarity to FER, can also directly bind with the FLS2-BAK1 complex but negatively regulate PTI (Mang et al., 2017). Soybean (Glycine max) also harbours a similar module, the malectin-like receptor kinase GmLMM1, that serves as a molecular adjustor in regulating immune activation . These findings illustrated that FER manages diverse cellular processes in response to different pathogens, but little is known about how FER works against maize fungal diseases.
To further understand the effect of FERONIA-like receptor genes in the response to multiple diseases in maize, we characterized three AtFER homologues: ZmFLR1 (Zm00001d047533), ZmFLR2 (Zm00001d029047), and ZmFLR3 (Zm00001d002175). All three maize proteins were membrane localized and were able to cause plant cell death. Furthermore, we generated virus-induced gene si-  (Table S2). These three highly homologous maize FLRs share a common structure with AtFER with an amino-terminal MLD and a carboxy-terminal intracellular serine/ threonine kinase domain ( Figure S1a,b). The expression profile of the 14 maize putative CrRLK1Ls was analysed by using published maize GSE27004 data (PRJNA137659) (Sekhon et al., 2011). The mRNA expression patterns of ZmFLR1 and ZmFLR2 were very similar, with highest expression in silks and the pericarp, while ZmFLR3 was expressed in all tissues except the embryo ( Figure S2).

F I G U R E 1
Phylogenetic tree of the CrRLK1L family proteins. Full-length amino acid sequences of 62 FER homologues from 35 diverse plant species were used to construct the phylogenetic tree via the neighbour-joining method with 1000 bootstrap values in MEGA 7 and was optimized with the iTOL online tool. The analysed CrRLK1Ls were classified into five subgroups, I-V, marked with different background colours. AtFER is highlighted in red and ZmFLR1, ZmFLR2, and ZmFLR3 are highlighted in green (Zm, Zea mays).

| ZmFLRs induce cell death in N. benthamiana leaves
To determine the function of ZmFLRs, we transiently overexpressed their coding sequences (CDSs), ECD, and serine/threonine kinase domain in N. benthamiana. We employed BAX as the positive control, which is able to trigger a strong cell death when expressed in tobacco (Lacomme & Santa Cruz, 1999 for the induction of cell death ( Figure 3). We also coexpressed ZmFLRs with LUC in maize protoplast to detect cell death. We found that ZmFLR1 and ZmFLR2 induced cell death more rapidly than ZmFLR3 when incubated for 12 h ( Figure S3). These results revealed that ZmFLRs may act as positive regulators of plant cell death.

| ZmFLRs are required for the ROS burst triggered by flg22 and chitin
Based on the cell death phenotype that ZmFLRs could trigger in N. benthamiana leaves and their differential expression following pathogen inoculation, we were interested in the function of

| ZmFLRs confer resistance to multiple pathogens
To further investigate the character of ZmFLRs in the resistance to major foliar fungal diseases in maize, we assessed the resist- The expression levels of ZmPR3 and ZmPR4 were similar to ZmPR1 and ZmPR5 in response to all four pathogens ( Figure S4).

| DISCUSS ION
The members of the CrRLK1L subfamily exist specifically and ex-  first discovered to have Mn 2+ -dependent serine/threonine protein kinase activity but, unlike other RLKs members, the kinase activity of CrRLK1 is achieved through intramolecular rather than intermolecular autophosphorylation (Schulze-Muth et al., 1996). The serine/ threonine kinase domain of FER has been shown to have autophosphorylation activity in an in vitro kinase assay (Kessler et al., 2015).
Although CrRLK1L is a small receptor-like protein kinase subfamily in plants, it has been shown to be extensively expressed in different plant tissues (Franck et al., 2018). Increasing evidence indicates that CrRLK1L members probably use varied motifs to interact with diverse ligands or signal molecules in different tissues, organs, or developmental stages (Franck et al., 2018).  (Figure 5c,d). Taken together, our data suggest that ZmFLRs may positively regulate PTI. This conclusion is consistent with the observation that AtFER probably serves as a scaffold protein to promote the ligand-induced FLS2-BAK1 and ERF-BAK1 interactions (Stegmann et al., 2017).
The other two members of the CrRLK1L family, ANX1 and ANX2, interact with FLS2 to negatively regulate FLS2-mediated antibacterial immunity, possibly by inducing segregation of BAK1 (Mang et al., 2017). GmLMM1 can restrain the FLS2-BAK1 sequestration with flg22 treatment, and it serves as a molecular adjustor in regulating immune activation by controlling the FLS2-BAK1 interaction   (Loake & Grant, 2007;Van Loon et al., 2006).
When challenged with fungal pathogens, fer mutant plants were more resistant to G. orontii, F. oxysporum, and M. oryzae (Kessler et al., 2010;Masachis et al., 2016). Similarly, mutants of the FER homologous genes Osflr2, Osflr11, and Gmlmm1 also have enhanced resistance to M. oryzae and oomycete pathogens Yang et al., 2020). Nevertheless, this research does not sufficiently indicate that FER negatively regulates immunity in this circumstance, but rather that FER and its dependent signalling pathways are frequently targeted by pathogenic fungi (Franck et al., 2018). In our current study, ZmFLRs conferred enhanced resistance to S. turcica, B. zeicola, C. graminicola, and B. maydis. The penetrates the host surface through mechanical pressure and enzymatic hydrolysis to form biotrophic hyphae, which inhibit plant immunity and obtain nutrients from living cells. Later, these fungi switch to a necrotrophic phase in which rapidly growing hyphae kill and destroy host tissues (Kleemann et al., 2012;Liu et al., 2015;Wang et al., 2021). SLB is caused by the necrotrophic fungus B. maydis. Necrotrophs are plant pathogens that degrade plant components or kill the plant by secreting lytic enzymes or toxins. Subsequently, the pathogen acquires nutrients from dead or dying tissues (Mayer et al., 2001;Shao et al., 2021).

| Identification of FLR genes in maize
The CDS and protein sequence data of maize B73 (Z. mays) were downloaded from the Maize Genetics and Genomics Database (Maize GDB: https://maize gdb.org/). Sixteen FLRs of Oryza sativa japonica rice

| Pathogen inoculation assay
For S. turcica, the spore suspension was sprayed onto the maize VIGS plants at a concentration of 10 5 spores/ml and samples were taken at 0, 12, 24, 36, 48, 60, and 72 hpi to detect the PR-protein gene expression. The lesion width was measured at 10 dpi inoculation with ImageJ software. The average lesion width was calculated from at least 20 randomly selected lesions . For B. zeicola, C. graminicola, and B. maydis, the spore suspensions were sprayed on the maize VIGS plants at a concentration of 10 5 spores/ml and samples were taken at 0, 12, 24, 36, 48, 60, and 72 hpi to detect the PRprotein gene expression. For the pathogen quantification, the fourth maize leaf was detached, placed in a petri dish (25 × 25 cm) containing wet filter paper, and inoculated with a spore suspension of 10 5 spores/ml. Inoculated leaves were cultured in a chamber at 95% humidity. Leaves were sampled at 5 dpi from the fourth leaf with about the same area. All primers used for VIGS plasmid construction and pathogen quantification are listed in Table S1. Photographs of diseased maize leaves were taken and the lesion areas were calculated by using ImageJ.

| ROS production assay
At 14 days after FoMV inoculation, a minimum of 30 leaf discs was taken from the five plants with a 4-mm diameter puncher. The leaf discs were incubated in 20 ml of sterile water on a 9-cm petri dish overnight in darkness. Then the leaf discs were transferred to 1.5-ml tubes containing 100 μl of luminol (Bio-Rad Immun-Star horseradish peroxidase substrate), 1 μl of horseradish peroxidase (HRP), and 1 μl of 1 mM flg22 or 1 μl of 0.8 mM chitin. The signal was then immediately collected using a Glomax20/20luminometer (Promega) every minute for a total of 20 min. Three biological replicates were assayed for each sample.

| Transient expression of ZmFLRs in N. benthamiana
ZmFLR1, ZmFLR2, and ZmFLR3 were cloned into the pCAMBIA1300-GFP vector and then these plasmid constructs were introduced into A. tumefaciens EHA105 using the electroporation method. For subcellular localization of ZmFLR1, ZmFLR2, or ZmFLR3 in N. benthamiana leaves, the cells were harvested and resuspended in an infiltration buffer (10 mM MES pH 5.6, 10 mM MgCl 2 , 200 μM acetosyringone) to an OD 600 of 1.0. The suspensions were infiltrated into 6-week-old N. benthamiana leaves (Lee et al., 2009). At 2 days after infiltration, the fluorescence was detected with a confocal microscope (LSM 980; Zeiss).
For ZmFLRs-induced cell death, A. tumefaciens cells carrying BAX and ZmFLRs were collected and resuspended to a final OD 600 of 0.2 and 1.0 with infiltration buffer, respectively. ZmFLRs, ZmFLRs ECD or ZmFLRs KD and BAX were infiltrated into the same N. benthamiana leaves. A. tumefaciens cells carrying eGFP were infiltrated as a negative control. The cell death phenotypes were analysed 4 days after transient expression. The leaves were cleared in boiling ethanol for 10 min until the chlorophyll was completely removed and then photographed. Each assay had at least three biological replicates.

| Transient expression of ZmFLRs in maize protoplast
Maize protoplasts were isolated from 10-day-old etiolated seedlings according to the method described previously (Yu et al., 2021). Then 5 μg of pCAMBIA1300-GFP-ZmFLRs and pRTV-myc-LUC was coexpressed in 250 μl of maize protoplasts. After 12 h of incubation in the dark at room temperature, 1 mM D-luceferin (Biovison) was mixed with the resuspended protoplasts and the luminescence signal from each sample was collected using a GloMax 96 microplate luminometer (Promega).

| Statistics analysis
The data were statistically analysed using Prism v. 7.00 (GraphPad Software Inc.). Dunnett's test was calculated for multiple comparisons, and Student's unpaired t test was used for pairwise comparisons. p values <0.05 were considered significant.

ACK N OWLED G EM ENTS
This work was financially supported by grants from the National

DATA AVA I L A B I L I T Y S TAT E M E N T
The sequences are available at GenBank (https://www.ncbi.nlm.nih.
gov/genba nk/ as accession numbers ZmFLR1: AQL06862; ZmFLR2: ONL97729; ZmFLR3: ONM13343. Other data that support the finding of this study are available from the corresponding author upon reasonable request.