The Receptor-like Kinase TaCRK-7A Inhibits Fusarium pseudograminearum Growth and Mediates Resistance to Fusarium Crown Rot in Wheat

Simple Summary Fusarium crown rot (FCR), caused by a soil-borne fungus Fusarium pseudograminearum, is one of the most destructive diseases of cereal crops, including wheat (Triticum aestivum) in many countries. It is vital to isolate resistance genes for improving crop resistance. Herein, we report the positive function of the wheat DUF26 domain-containing receptor-like kinase TaCRK-7A in the host resistance response to the pathogen F. pseudograminearum attack. The purified TaCRK-7A protein directly inhibited F. pseudograminearum mycelial growth. The TaCRK-7A transcript was elevated upon F. pseudograminearum infection and the transcript induction was higher in resistant wheat genotypes than in susceptible wheat genotypes. Knocking down of TaCRK-7A compromised resistance of wheat to FCR and significantly reduced the transcript levels of defense genes in wheat. This study provides a novel insight into the wheat immune responses to F. pseudograminearum. Abstract The fungus F. pseudograminearum can cause the destructive disease Fusarium crown rot (FCR) of wheat, an important staple crop. Functional roles of FCR resistance genes in wheat are largely unknown. In the current research, we characterized the antifungal activity and positive-regulatory function of the cysteine-rich repeat receptor-like kinase TaCRK-7A in the defense against F. pseudograminearum in wheat. Antifungal assays showed that the purified TaCRK-7A protein inhibited the growth of F. pseudograminearum. TaCRK-7A transcript abundance was elevated after F. pseudograminearum attack and was positively related to FCR-resistance levels of wheat cultivars. Intriguingly, knocking down of TaCRK-7A transcript increased susceptibility of wheat to FCR and decreased transcript levels of defense-marker genes in wheat. Furthermore, the transcript abundances of TaCRK-7A and its modulated-defense genes were responsive to exogenous jasmonate treatment. Taken together, these results suggest that TaCRK-7A can directly inhibit F. pseudograminearum growth and mediates FCR-resistance by promoting the expression of wheat defense genes in the jasmonate pathway. Thus, TaCRK-7A is a potential gene resource in FCR-resistant wheat breeding program.


Introduction
Fusarium crown rot (FCR) is a destructive disease of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) worldwide [1]. It causes serious yield loss [2]. The soilborne fungus Fusarium pseudograminearum is a major causal pathogen responsible for FCR Biology 2021, 10 disease [1][2][3]. Usage of resistant wheat cultivars is one effective way to control FCR in wheat. It is crucial to isolate the resistance genes for molecular breeding wheat with FCR resistance. Several groups already mapped wheat QTLs conferring resistance to FCR, including the QTLs on 3B, 6A, 4D, and 7A [1,[4][5][6]. Most recently, a paper reported the wheat dirigent gene TaDIR-B1 with negative role, whose loss-of-function enhances FCRresistance in wheat [7]. However, functional roles of FCR resistance genes in wheat are largely unknown. In Arabidopsis and crop plants, several cysteine-rich repeat receptor-like protein kinases (CRKs) have been shown to confer resistance or be involved in the plant innate immune responses to bacterial and fungal pathogens [8][9][10][11][12]. The protein sequences of CRKs all include an intracellular serine/threonine protein kinase domain, a transmembrane domain, and two copies of the extracellular Domain 26 of Unknown Function (DUF26, antifungal domain) each with a plant specific cysteine-rich motif [9,11]. The secreted ginkbilobin2 (Gnk2) protein from gymnosperm Ginkgo biloba comprises a single DUF26 domain and has been demonstrated to have antifungal activity [13]. The fungal mannose binding of three residues (asparagine-11, arginine-93, and glutamate-104) in its DUF26 domain has been determined to be necessary for the antifungal activity of Gnk2 [13,14]. In our previous study, a wheat CRK protein TaCRK3 was shown to defend against the wheat sharp eyespot pathogen Rhizoctonia cerealis [15]. The gene sequence is matched with the sequence TraesCS7A02G105100.1 and thus was named TaCRK-7A. However, no study about the functional roles of wheat CRK proteins in host resistance responses to F. pseudograminearum has been reported yet.
In this current report, we examined in vitro inhibition activity of the purified TaCRK-7A protein against the growth of F. pseudograminearum and investigated its functional role in the wheat resistance to FCR caused by F. pseudograminearum infection.
The pathogenic fungus F. pseudograminearum strain WHF220 was isolated from the FCR-symptomatic wheat sheaths in Shandong by Prof. Jinfeng Yu and Dr. Li Zhang (Shandong Agricultural University, Tai'an, China).
To investigate the transcript profile and defensive role of TaCRK-7A, wheat plants at the early-tiller stage were inoculated with toothpicks harboring the well-developed mycelia of F. pseudograminearum. Furthermore, seedlings of wheat cultivar CI12633 at three-leaf stage were treated with 0.05 mM methyl jasmonate (MeJA, a jasmonate analog) and sampled after spraying for 0.5, 1, 3, 6, 12, and 24 h as described by Zhang et al. [18].

Assay on Inhibition of Purified TaCRK-7A against F. pseudograminearum Mycelial Growth
According to the protocol described previously [15], the His-TF-TaCRK-7A recombinant or His-TF proteins were separately expressed in Escherichia coli DE3 cells, secreted into the culture supernatant, and purified. The purified His-TF-TaCRK-7A or His-TF control proteins were separately added into the middle pore of the potato dextrose agar (PDA) medium plate. Subsequently, the liquid mycelia of F. pseudograminearum was inoculated into the each pore and subjected to further incubation at 25 • C for 5 days [15]. Photographs for F. pseudograminearum mycelia/hyphae growth status were taken on the fifth day. These assays were repeated three times.

Assesment on Defense Role of TaCRK-7A in Wheat against FCR
By means of the barley yellow dwarf virus (BSMV)-based virus-induced gene silencing (VIGS) experiments as described [19], the BSMV:TaCRK-7A VIGS construct was prepared [15] and the TaCRK-7A transcript in the resistant wheat cultivar (cv.) CI12633 Biology 2021, 10, 1122 3 of 10 plants was knocked down. Twenty days after the virus transfection, TaCRK-7A-silenced or the BSMV:GFP-infected (control) wheat plants were further inoculated with F. pseudograminearum strain WHF220 as previously described [7]. At~28 d post inoculation (dpi) with the fungal pathogen, the average lesion length and width of FCR on the inoculated sheaths of these plants were measured, and the infection types of these wheat plants were rated [1,17]. In addition, Trypan blue staining for F. pseudograminearum hyphae was used to assess the defense role of TaCRK-7A following Zhang et al. 2012 [18]. The experiments were performed two batches. At least 15 plants for each construct were tested in each batch.

RNA Extraction and Real-Time Quantitative PCR (qRT-PCR) Analysis
The extraction of RNAs from wheat samples was conducted by Trizol reagent (Invitrogen, Carlsbad, CA, USA). Then, the RNA was purified and was reverse-transcribed into cDNA by using the FastQuant RT Kit (Tiangen, Beijing, China). The transcript levels of TaCRK-7A and four defense-marker genes in wheat plants were tested by using the gene-specific primers [15] and the qRT-PCR technique [15,18,19]. The qRT-PCR was conducted by using SYBR Premix Ex Taq kit (TaKaRa, Otsu, Japan) on an ABI 7500 instrument (Applied Biosystems, Waltham, MA, USA). The relative transcript levels of the tested genes were calculated with the 2 −∆∆CT method [20].

The Purified TaCRK-7A Protein Directly Inhibits F. pseudograminearum Hyphae Growth
The deduced TaCRK-7A protein sequence includes an intracellular serine/threonine protein kinase domain, a transmembrane domain, and two DUF26 domains with the three conversed amino acids (asparagine, arginine, and glutamate) that are required for mannose binding activity and for antifungal activity [13]. We performed in vitro antifungal assay as described by Guo et al. 2021 [15]. The antifungal assay results showed that compared to the His-TF tag protein from the original vector pCOLD, the purified His-TF-TaCRK-7A protein could obviously inhibit the mycelial/hyphae growth of F. pseudograminearum strain WHF220 ( Figure 1A,B). These data suggested that TaCRK-7A protein possesses antifungal activity against F. pseudograminearum. The activity is possibly due to the three conversed amino acids (asparagine, arginine, and glutamate) in the extracellular DUF26 domain. In our previous study, the purified DUF26 domain in TaCRK-7A, like the whole protein, could inhibit the mycelial/hyphae growth of R. cerealis, indicating that the DUF26 domain is required for the antifungal activity of TaCRK-7A protein [15]. Similarly, previous papers reported that the DUF26 domain-containing peptide Gnk2 in G. biloba, due to binding with mannose of fungal wall cells, could inhibit the growth of several plant-pathogenic fungi including Fusarium species [13,14]. Meanwhile, the maize DUF26-domain-containing peptide ZmAFP1 binds fungal mannose and displays antifungal activity only against the repetitive effector-coding gene rsp3 mutant of the biotrophic fungus Ustilago maydis, but not against the fungal strain constitutively expressing rsp3 [21].

TaCRK-7A Transcript Abundance Is Related to FCR-Resistance Degree of Wheat Accessions
TaCRK-7A promoter sequence includes two TC-rich cis-acting elements that are reported to be involved in defense and stress responses [15]. Our qRT-PCR analyses showed that in comparison with non-inoculation, TaCRK-7A transcript abundance was significantly elevated in wheat after F. pseudograminearum attack and reached a peak at 4 dpi with the pathogen (Figure 2A), consistent with the promoter sequence characteristic. Intriguingly, the gene expression level at 4 dpi was related to the FCR-resistance degree of the wheat cultivars tested ( Figure 2B). These data imply that TaCRK-7A might be involved in wheat resistance against FCR. To further explore the cause of the transcript difference, we cloned and sequenced the TaCRK-7A sequences from these six wheat cultivars. As a result, there was no genetic variation within the promoter and most gene-body sequences, but a single nucleotide polymorphism (SNP) existed in the third intron of the gene. Namely, a nucleotide "A" at position 1686-bp in two resistant wheat cultivars (Chinese spring and CI12633) was replaced by "G" at the site in the susceptible varieties Yangmai6, Jimai22, and Yangmai 158, as well as in the resistant cultivar Nivat 14 ( Figure S1). Therefore, we supposed that the TaCRK-7A transcript difference between resistant and susceptible wheat cultivars might be associated with epigenetic events. Similarly, epigenetic events contributed to the transcription of TaCYP81D5 conferring salinity tolerance, whereas no genetic variation within the promoter and the genebody sequences of TaCYP81D5 appeared between tolerant and intolerant wheat cultivars [22].
Further sequence search indicated that in wheat genome, there is a paralogous gene TaCRK-7D with sequence identity number TraesCS7D02G099300.1. The TaCRK-7D protein also contains two DUF26 domains, and has 82.58% identity in amino aide sequence of TaCRK-7A protein. TaCRK-7D gene shares 89.70% identity with TaCRK-7A in nucleo-

TaCRK-7A Transcript Abundance Is Related to FCR-Resistance Degree of Wheat Accessions
TaCRK-7A promoter sequence includes two TC-rich cis-acting elements that are reported to be involved in defense and stress responses [15]. Our qRT-PCR analyses showed that in comparison with non-inoculation, TaCRK-7A transcript abundance was significantly elevated in wheat after F. pseudograminearum attack and reached a peak at 4 dpi with the pathogen (Figure 2A), consistent with the promoter sequence characteristic. Intriguingly, the gene expression level at 4 dpi was related to the FCR-resistance degree of the wheat cultivars tested ( Figure 2B). These data imply that TaCRK-7A might be involved in wheat resistance against FCR. To further explore the cause of the transcript difference, we cloned and sequenced the TaCRK-7A sequences from these six wheat cultivars. As a result, there was no genetic variation within the promoter and most gene-body sequences, but a single nucleotide polymorphism (SNP) existed in the third intron of the gene. Namely, a nucleotide "A" at position 1686-bp in two resistant wheat cultivars (Chinese spring and CI12633) was replaced by "G" at the site in the susceptible varieties Yangmai6, Jimai22, and Yangmai 158, as well as in the resistant cultivar Nivat 14 ( Figure S1). Therefore, we supposed that the TaCRK-7A transcript difference between resistant and susceptible wheat cultivars might be associated with epigenetic events. Similarly, epigenetic events contributed to the transcription of TaCYP81D5 conferring salinity tolerance, whereas no genetic variation within the promoter and the genebody sequences of TaCYP81D5 appeared between tolerant and intolerant wheat cultivars [22]. tide sequence. In fact, the qRT-PCR experiment showed that the transcript of TaCRK-7D was also induced by F. pseudograminearum infection ( Figure S2). It is very interesting to investigate the effect of TaCRK-7D in the wheat resistance response to FCR in future.  TaCRK-7A in six wheat cultivars at 4 dpi with F. pseudograminearum WHF220. The gene transcript in the highly susceptible wheat cultivar Yangmai 158 was set to 1. R represents resistant wheat cv.; MR represents moderately resistant wheat cultivars; MS represents moderately susceptible wheat cultivars; S represents highly susceptible wheat cv.. TaActin was used as the internal control for gene expression. ** p < 0.01 (Student's t-test) represents significant differences derived from three repeats. Bars indicate the standard deviation of the mean.

TaCRK-7A Is Required for Resistance of Wheat to FCR
To assess the function of TaCRK-7A in wheat defense against FCR caused by infection of F. pseudograminearum, we performed two batches of the functional assays in the mildly resistant wheat cv. CI12633 by means of VIGS plus disease evaluation. qRT-PCR assay showed that, at the virus transfection for 12 d, the TaCRK-7A transcript level was markedly decreased in BSMV:TaCRK-7A-transfected CI12633 plants compared to BSMV:GFP-infectedCI12633 (control) plants ( Figure 3A). These TaCRK-7A-silenced and control plants were further inoculated with F. pseudograminearum strain WHF220. At 7 dpi with the fungal pathogen, microscopic observation indicated that more F. pseudograminearum hyphae appeared on the fungus-infected sheaths of BSMV:TaCRK-7A-silenced wheat plants than on those of BSMV:GFP-infected plants ( Figure 3B). The FCR disease assessments showed that in comparison with BSMV:GFP-infected plants, TaCRK-7A-silenced plants displayed more susceptibility to FCR caused by F. pseudograminearum infection, such as necrosis length and disease se- TaCRK-7A transcript level at none is set to 1. (B) Transcript patterns of TaCRK-7A in six wheat cultivars at 4 dpi with F. pseudograminearum WHF220. The gene transcript in the highly susceptible wheat cultivar Yangmai 158 was set to 1. R represents resistant wheat cv.; MR represents moderately resistant wheat cultivars; MS represents moderately susceptible wheat cultivars; S represents highly susceptible wheat cv.. TaActin was used as the internal control for gene expression. ** p < 0.01 (Student's t-test) represents significant differences derived from three repeats. Bars indicate the standard deviation of the mean.
Further sequence search indicated that in wheat genome, there is a paralogous gene TaCRK-7D with sequence identity number TraesCS7D02G099300.1. The TaCRK-7D protein also contains two DUF26 domains, and has 82.58% identity in amino aide sequence of TaCRK-7A protein. TaCRK-7D gene shares 89.70% identity with TaCRK-7A in nucleotide sequence. In fact, the qRT-PCR experiment showed that the transcript of TaCRK-7D was also induced by F. pseudograminearum infection ( Figure S2). It is very interesting to investigate the effect of TaCRK-7D in the wheat resistance response to FCR in future.

TaCRK-7A Is Required for Resistance of Wheat to FCR
To assess the function of TaCRK-7A in wheat defense against FCR caused by infection of F. pseudograminearum, we performed two batches of the functional assays in the mildly resistant wheat cv. CI12633 by means of VIGS plus disease evaluation. qRT-PCR assay showed that, at the virus transfection for 12 d, the TaCRK-7A transcript level was markedly decreased in BSMV:TaCRK-7A-transfected CI12633 plants compared to BSMV:GFP-infectedCI12633 (control) plants ( Figure 3A). These TaCRK-7A-silenced and control plants were further inoculated with F. pseudograminearum strain WHF220. At 7 dpi with the fungal pathogen, microscopic observation indicated that more F. pseudogramin-earum hyphae appeared on the fungus-infected sheaths of BSMV:TaCRK-7A-silenced wheat plants than on those of BSMV:GFP-infected plants ( Figure 3B). The FCR disease assessments showed that in comparison with BSMV:GFP-infected plants, TaCRK-7A-silenced plants displayed more susceptibility to FCR caused by F. pseudograminearum infection, such as necrosis length and disease severity ( Figure 3C,D). In two batch experiments, the average infection types (ITs) on stems of TaCRK-7A-silenced plants were 4.09 ± 1.00 and 4.70 ± 0.71, whereas those for BSMV:GFP-transfected plants were 2.20 ± 0.87 and 3.85 ± 0.96. These data pointed to the conclusion that the functional TaCRK-7A, acting as a positive regulator, was required for wheat resistance to FCR caused by infection of F. pseudograminearum. The above results indicated TaCRK-7A transcript was induced by F. pseudograminearum from 1 to 7 d, and the transcripts in six wheat cultivars at 4 dpi were correlated with the resistance levels of the tested cultivars ( Figure 2). Based on the previous experiences [7,15,19], it was deduced that the TaCRK-7A transcript at 7 dpi might be also related with the disease resistance. To ensure enough development of FCR disease in the VIGS plus the fungal pathogen experiments, we sampled these wheat sheaths inoculated with the fungus at 7 d for further hyphae staining and qRT-PCR analysis.
Biology 2021, 10, x FOR PEER REVIEW 6 of 10 verity ( Figure 3C,D). In two batch experiments, the average infection types (ITs) on stems of TaCRK-7A-silenced plants were 4.09 ± 1.00 and 4.70 ± 0.71, whereas those for BSMV:GFP-transfected plants were 2.20 ± 0.87 and 3.85 ± 0.96. These data pointed to the conclusion that the functional TaCRK-7A, acting as a positive regulator, was required for wheat resistance to FCR caused by infection of F. pseudograminearum. The above results indicated TaCRK-7A transcript was induced by F. pseudograminearum from 1 to 7 d, and the transcripts in six wheat cultivars at 4 dpi were correlated with the resistance levels of the tested cultivars ( Figure 2). Based on the previous experiences [7,15,19], it was deduced that the TaCRK-7A transcript at 7 dpi might be also related with the disease resistance. To ensure enough development of FCR disease in the VIGS plus the fungal pathogen experiments, we sampled these wheat sheaths inoculated with the fungus at 7 d for further hyphae staining and qRT-PCR analysis.

TaCRK-7A and Its Modulated-Defense Genes Were Responsive to JA Stimulus
To explore the molecular mechanism underlying the FCR-resistance function of TaCRK-7A, we first examined if TaCRK-7A modulates the expression of wheat defense genes. These experiments showed that knocking-down of TaCRK-7A significantly decreased the transcript levels of four defense-marker genes in TaCRK-7A-silenced wheat plants at 7 dpi with F. pseudograminearum WHF220 (Figure 4). The tested wheat defense-marker genes include PR2 (TaGluD), chitinase 1 (TaChit1), chitinase 3 (TaChit3), and chitinase 4 (TaChit4). The data suggested that the functional TaCRK-7A might be upstream of these defense-marker genes and also up-regulated their expression levels during wheat resistance responses against F. pseudograminearum infection. The results support previous reports about CRKs acting as upstream regulatory factors [10,15].

TaCRK-7A and Its Modulated-Defense Genes Were Responsive to JA Stimulus
To explore the molecular mechanism underlying the FCR-resistance function of TaCRK-7A, we first examined if TaCRK-7A modulates the expression of wheat defense genes. These experiments showed that knocking-down of TaCRK-7A significantly decreased the transcript levels of four defense-marker genes in TaCRK-7A-silenced wheat plants at 7 dpi with F. pseudograminearum WHF220 (Figure 4). The tested wheat defense-Biology 2021, 10, 1122 7 of 10 marker genes include PR2 (TaGluD), chitinase 1 (TaChit1), chitinase 3 (TaChit3), and chitinase 4 (TaChit4). The data suggested that the functional TaCRK-7A might be upstream of these defense-marker genes and also up-regulated their expression levels during wheat resistance responses against F. pseudograminearum infection. The results support previous reports about CRKs acting as upstream regulatory factors [10,15]. Furthermore, TaCRK-7A promoter contains jasmonate (JA)-responsive cis-acting elements (CGTCA-motif and TGACG-motif). Thus, we analyzed the transcript profile of TaCRK-7A in the wheat cv. CI12633 plants treated by external JA for 0.5, 1, 3, 6, 12, and 24 h. The analysis showed that compared to non-treatment (none), TaCRK-7A transcript level was significantly increased after JA treatment from 0.5 h to 24 h, and the induction reached the first peak at 6 h ( Figure 5A). Moreover, the promoter sequences of TaChit1, TaChit3, TaChit4, and TaGluD, whose transcripts were positively modulated by TaCRK-7A (Figure 4), all contain JA-responsive cis-acting elements. Thus, transcript profiles of these defense-marker genes were tested in the wheat cv. CI12633 plants treated with JA for 0.5 h and 6 h. As a result, compared with the mock treatment, transcript levels of TaChit1, TaChit3, TaChit4, and TaGluD were significantly increased after JA treatment ( Figure 5B). These results suggest that TaCRK-7A and its modulated-defense genes (TaChit1, TaChit3, TaChit4, and TaGluD) were all responsive to the JA stimulus. Similarly, a previous paper reported that GbCRK18, a cotton CRK-encoding gene, was also responsive to exogenous JA stimulus [23]. Taken together, these results suggest that TaCRK-7A mediates wheat resistance to FCR through promoting the expression of four wheat defense-marker genes in JA signaling. Silencing of TaCRK-7A reduced transcript abundances of defense genes in wheat CI12633 plants at 7 dpi with F. pseudograminearum WHF220. Relative transcript abundances of these defense genes in BSMV:TaCRK-7A-silenced CI12633 plants were quantified relative to those in BSMV:GFPinfected control plants. TaActin was used as internal control. Statistically significant differences were determined based on three replications using Student's t-test (* p < 0.05, ** p < 0.01).
Furthermore, TaCRK-7A promoter contains jasmonate (JA)-responsive cis-acting elements (CGTCA-motif and TGACG-motif). Thus, we analyzed the transcript profile of TaCRK-7A in the wheat cv. CI12633 plants treated by external JA for 0.5, 1, 3, 6, 12, and 24 h. The analysis showed that compared to non-treatment (none), TaCRK-7A transcript level was significantly increased after JA treatment from 0.5 h to 24 h, and the induction reached the first peak at 6 h ( Figure 5A). Moreover, the promoter sequences of TaChit1, TaChit3, TaChit4, and TaGluD, whose transcripts were positively modulated by TaCRK-7A (Figure 4), all contain JA-responsive cis-acting elements. Thus, transcript profiles of these defense-marker genes were tested in the wheat cv. CI12633 plants treated with JA for 0.5 h and 6 h. As a result, compared with the mock treatment, transcript levels of TaChit1, TaChit3, TaChit4, and TaGluD were significantly increased after JA treatment ( Figure 5B). These results suggest that TaCRK-7A and its modulated-defense genes (TaChit1, TaChit3, TaChit4, and TaGluD) were all responsive to the JA stimulus. Similarly, a previous paper reported that GbCRK18, a cotton CRK-encoding gene, was also responsive to exogenous JA stimulus [23]. Taken together, these results suggest that TaCRK-7A mediates wheat resistance to FCR through promoting the expression of four wheat defense-marker genes in JA signaling.

Conclusions
The current research revealed a novel defense role of the wheat RLK TaCRK-7A. TaCRK-7A not only directly inhibits the hyphae growth of F. pseudograminearum, but also mediates wheat resistance to FCR through promoting the expression of wheat defense-marker genes in JA signaling pathway. This study sheds a light on the molecular mechanisms underlying wheat defense against F. pseudograminearum and deepens understanding of the mechanisms of CRKs in plant-fungi interactions. Practically, TaCRK-7A is a promising gene for molecular breeding wheat with resistance to FCR caused by F. pseudograminearum.

Conclusions
The current research revealed a novel defense role of the wheat RLK TaCRK-7A. TaCRK-7A not only directly inhibits the hyphae growth of F. pseudograminearum, but also mediates wheat resistance to FCR through promoting the expression of wheat defensemarker genes in JA signaling pathway. This study sheds a light on the molecular mechanisms underlying wheat defense against F. pseudograminearum and deepens understanding of the mechanisms of CRKs in plant-fungi interactions. Practically, TaCRK-7A is a promising gene for molecular breeding wheat with resistance to FCR caused by F. pseudograminearum.