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. 2019 Apr 11;180(2):1219–1229. doi: 10.1104/pp.18.01328

Hypermorphic SERK1 Mutations Function via a SOBIR1 Pathway to Activate Floral Abscission Signaling

Isaiah Taylor a,b,c,d, John Baer a,b,e, Ryan Calcutt a,b,f, John C Walker a,b,1,2
PMCID: PMC6548279  PMID: 30975695

Gain of function mutations in SERK1 suppress the abscission defect of the haesa/haesa-like 2-mutant by activating a SOBIR1 signaling pathway

Abstract

In Arabidopsis (Arabidopsis thaliana), the abscission of floral organs is regulated by two related receptor-like protein kinases, HAESA (HAE) and HAESA-LIKE2 (HSL2). In complex with members of the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family of coreceptor protein kinases, HAE and HSL2 are activated when bound by INFLORESCENCE DEFICIENT IN ABSICSSION, a proteolytically processed peptide ligand, activating the expression of genes encoding secreted cell wall remodeling and hydrolase enzymes. hae hsl2 mutants fail to induce expression of these genes and retain floral organs indefinitely. Here, we report identification of an allelic series of hae hsl2 suppressor mutations in the SERK1 coreceptor protein kinase gene. Genetic and transcriptomic evidence indicates that these alleles represent a novel class of gain-of-function mutations that activate signaling independently of HAE/HSL2. We show that, surprisingly, the suppression effect does not rely on the protein kinase activity of SERK1 and that activation of signaling relies on the receptor-like kinase gene SUPPRESSOR OF BIR1 (SOBIR1). The effect of these mutations can be mimicked by loss of function of BAK1-INTERACTING RECEPTOR-LIKE KINASE1 (BIR1), a known negative regulator of SERK-SOBIR1 signaling. These results suggest that BIR1 negatively regulates SERK-SOBIR1 signaling during abscission and that the identified SERK1 mutations likely interfere with this negative regulation.

Abscission is the process by which plants shed structures, such as fruit, leaves, and floral organs. Abscission occurs as the result of a developmental process or is triggered by damage or adverse environmental conditions. In Arabidopsis (Arabidopsis thaliana), abscission of sepals, petals, and stamen follows pollination in a developmentally programmed manner, while abscission of cauline leaves occurs as a result of drought stress or pathogen infection (Patterson, 2001; Liljegren et al., 2009; Patharkar and Walker, 2016; Patharkar et al., 2017). Abscission is regulated by the two redundant Leu-rich repeat receptor-like protein kinases (RLKs), HAESA and HAESA-LIKE2 (HAE/HSL2; Jinn et al., 2000; Cho et al., 2008; Stenvik et al., 2008). Binding of the proteolytically processed, secreted peptide INFLORESCENCE-DEFICIENT IN ABSCISSION (IDA) induces the association of HAE/HSL2 and members of the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family of Leu-rich repeat RLK coreceptors (Meng et al., 2016; Santiago et al., 2016; Schardon et al., 2016). This association activates a downstream MAP kinase cascade composed of MAP kinase kinase 4 and 5 (MKK4/MKK5) and MAP kinase 3 and 6 (MPK3/MPK6; Cho et al., 2008; Meng et al., 2016). This MAP kinase cascade targets transcriptional repressors of the AGAMOUS-like family, including AGL15, leading to derepression of HAE expression and an increase in signaling via positive feedback (Fernandez et al., 2000; Adamczyk et al., 2007; Chen et al., 2011; Patharkar and Walker, 2015). This signaling pathway regulates expression of several genes involved in cell wall remodeling and hydrolysis of cell wall and middle lamella polymers such as pectin (Niederhuth et al., 2013b). Plants with mutations in HAE/HSL2 or IDA, or in high-order mutants of SERK genes, display floral abscission defects indicative of a failure to properly break down the middle lamella between the abscising organ and the body of the plant (Butenko et al., 2003; Cho et al., 2008; Meng et al., 2016). Additionally, mutants in the ADP-Ribosylation Factor GTPase Activating Protein gene NEVERSHED (NEV) are also abscission deficient and possess a disorganized secretory system in the abscission zone (Liljegren et al., 2009). The abscission-deficient phenotype of nev can be suppressed by mutations in SERK1 as well as mutations in the RLK gene SUPPRESSOR OF BIR1-1 (SOBIR1; also known as EVERSHED [EVR]) and the cytosolic RLK CAST AWAY (Leslie et al., 2010; Burr et al., 2011). The exact cause of the nev mutant phenotype, as well as the mechanism of nev suppression, are not fully understood. Recent work has demonstrated that nev mutants have ectopic lignification patterns in the abscission zone and exhibit widespread transcriptional reprogramming, including strong induction of biotic stress response gene expression (Lee et al., 2018; Taylor and Walker, 2018). These results together suggest that the nev phenotype may be related to misregulation of molecular signaling, possibly involving a pathway regulating lignification of the abscission zone.

In this work, we sought to understand additional regulators of abscission signaling using a genetic suppression approach. We identified an allelic series of putative gain-of-function SERK1 mutations capable of suppressing the abscission defect of hae hsl2. These mutant alleles represent a novel class of hypermorphic SERK gene mutations that appear to signal through the RLK SOBIR1, likely by interfering with the function of the negative regulator of signaling BAK1-INTERACTING RECEPTOR-LIKE KINASE1 (BIR1). These alleles provide insights into the regulation of downstream signaling processes by SERK proteins. Implications for understanding of the nev mutant are discussed.

RESULTS

Identification of hae-3 hsl2-3 Suppressor Mutations in the SERK1 Gene

To identify regulators of abscission signaling, we performed suppressor screens of the previously described hae-3 hsl2-3 abscission-deficient mutant (Niederhuth et al., 2013a, 2013b). We isolated three strong suppressors, an intermediate suppressor, and a weak suppressor. Initial mapping by sequencing of a strong suppressor identified a semidominant, linked missense mutation in the SERK1 gene, substituting Leu-326 for Phe (Fig. 1; Supplemental Table S1). Additional genetic analysis demonstrated that all five mutants contained semidominant, linked missense mutations affecting conserved SERK protein residues (Fig. 1; Supplemental Fig. S1; Supplemental Table S1).

Figure 1.

Figure 1.

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Phenotypes and molecular mapping of hae-3 hsl2-3 suppressor mutations. A, Floral abscission phenotypes of Columbia-0 (Col-0), hae-3 hsl2-3, and hae-3 hsl2-3 line 14.1. B, Landsberg erecta (Ler) single-nucleotide polymorphism (SNP) density resulting from mapping by sequencing of bulked DNA from strongly abscising hae hsl2 line 14.1 (Col-0) × hae hsl2 (Ler) F2 plants, indicating linkage to chromosome 1. C, Predicted location of hae hsl2 suppressor mutations on the SERK1 protein.

To gain insight into the possible functions of the mutated residues, we mapped them onto a model of the SERK1 intracellular domain based on the crystal structure of the closely related BAK1/SERK3 protein kinase domain as well as on the recently solved crystal structure of the SERK1 ectodomain (Yan et al., 2012; Santiago et al., 2013; Protein Data Bank IDs 3ULZ and 4LSC). The four protein kinase domain mutations are localized to a patch of three surface-exposed residues on the N-lobe of the protein kinase domain distal to the catalytic cleft (Fig. 1). The phenotypes of these mutants are all strong or intermediate (Supplemental Fig. S1; Supplemental Table S1). The extracellular domain mutation with the weak suppression phenotype maps to the ligand receptor-binding face of the ectodomain (Fig. 1). This residue, Arg-147, makes side chain interactions with the residue Asp-123, which is homologous to BAK1 residue Asp-122. Mutation of Asp-122 yields a hypermorphic variant of BAK1 displaying ligand-independent association with the brassinosteroid receptor BRI1 and reduced affinity for the ectodomain of members of the BIR family of negative regulators of signaling (Jaillais et al., 2011; Hohmann et al., 2018). However, we found the weak phenotype of this mutant difficult to study and instead focused on the intracellular domain mutants. Since the four strongest mutations cluster to a spatially restricted region of the protein kinase domain, we hypothesize that the mutations have a similar effect. To study this effect, we examined the serk1-L326F mutant as a representative allele.

Genetic Analysis of hae-3 hsl2-3 serk1-L326F

Because SERK1 positively regulates abscission and because these mutations are all semidominant, we hypothesized that they are gain-of-function mutations. We performed several genetic experiments to test this hypothesis. We first crossed the hae-3 hsl2-3 mutant with the well-characterized serk1-1 loss-of-function transfer DNA (T-DNA) insertion mutant. Consistent with previously published analysis of serk1 loss-of-function mutations (Lewis et al., 2010), we found that the serk1-1 allele is unable to suppress the abscission defect of hae-3 hsl2-3, demonstrating specificity of the suppression defect to the semidominant alleles isolated in our screen (Fig. 2). We next crossed the serk1-L326F mutant with the hae-1 hsl2-4 double mutant. This mutant has a T-DNA insertion in the first exon of HAE and a premature stop codon in the first exon of HSL2 and is a predicted protein null (Niederhuth et al., 2013a). We isolated the hae-1 hsl2-4 serk1-L326F triple mutant and found that it displays nearly complete suppression of the abscission defect similar to hae-3 hsl2-3 serk1-L326F, demonstrating nonspecificity of the suppression effect with regard to alleles of hae and hsl2 (Fig. 2). We further performed a transgenic recapitulation experiment, where we transformed SERK1pr::SERK1 wild-type and SERK1pr::SERK1-L326F mutant transgenes into hae-3 hsl2-3. In the T1 generation, we found that none of 16 plants transformed with the wild-type transgene displayed abscission (Fig. 2; Supplemental Table S2 for all transgene counts). For the SERK1pr::SERK1-L326F transgene, 10 of 30 T1 plants displayed weak or strong suppression (Fig. 2; Supplemental Table S2). These results are consistent with a model wherein serk1-L326F is a hypermorphic mutation.

Figure 2.

Figure 2.

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Genetic analysis of a representative hypermorphic SERK1 allele. A, Phenotype of the hae-3 hsl2-3 serk1-1 triple mutant. B, Phenotype of the hae-1 hsl2-4 serk1-L326F triple mutant. C, Transgenic recapitulation of the hae hsl2 suppression phenotype (T1 generation). D, Phenotypes of serk1 serk2 bak1 triple mutants. E, Phenotypes of HAEpr::YDA-YFP K429R transformed into Col-0 and hae-3 hsl2-3 serk1-L326F (T1 generation).

Next, we tested whether the serk1-L326F mutation interferes with the normal functions of SERK1. Prior work has shown that the serk1-1 serk2-1 double loss-of-function mutant is male sterile because of defective tapetum development and that the serk1-1 serk2-1 bak1-5 triple mutant is sterile and displays a weak abscission defect (Albrecht et al., 2005; Colcombet et al., 2005; Meng et al., 2016). We regenerated the serk1-1 serk2-1 bak1-5 triple mutant and confirmed the sterility and abscission-defective phenotypes (Fig. 2). We also created the serk1-L326F serk2-1 bak1-5 triple mutant and found that it displays wild-type fertility and abscises all its floral organs, indicating that the serk1-L326F mutant allele is a functional coreceptor in tapetum development and abscission (Fig. 2).

Next, we examined the effect of the serk1 suppressor mutations in relation to downstream signaling processes. Previously published MAP kinase signaling suppression strategies used in floral abscission research involve inefficient tandem RNA interference targeting MKK4/MKK5 or complicated MPK3/MPK6 transgenic/mutant combinations (Cho et al., 2008). Work in stomatal cellular identity specification has shown that MAP kinase signaling regulated by MKK4/MKK5-MPK3/MPK6 can be suppressed in a cell type-specific manner by expression of a kinase-inactive version of the MAPKKK YODA (YDA) gene (Lampard et al., 2009). The identity of the MAPKKK(s) upstream of MKK4/MKK5 during floral abscission is unknown. Nonetheless, we hypothesized that expression of kinase-inactive YDA under the control of the HAE promoter may block signaling by MKK4/MKK5 during floral abscission.

To test this approach, we created a HAEpr::YDA-YFP K429R construct designed to express a YDA-YFP fusion with a mutation of a conserved, catalytic Lys specifically in the abscission zone. We found that 33 of 64 T1 plants transformed with this construct in the Col-0 background displayed abscission defects similar to hae hsl2 mutants (Fig. 2; Supplemental Table S2). This phenotype is associated with YFP signal restricted to the abscission zone, where HAE is normally expressed (Fig. 2). We next tested the ability of this construct to block the suppression effect of hae-3 hsl2-3 serk1-L326F. We found that eight of 14 T1 hae-3 hsl2-3 serk1-L326F HAEpr::YDA-YFP K429R plants had abscission defects similar to hae hsl2 mutants (Fig. 2; Supplemental Table S2). These results indicate that the suppression effect acts upstream of MAP kinase signaling, which is consistent with our hypothesis of hypermorphic activation of signaling at the plasma membrane. These results also provide circumstantial evidence that YDA acts downstream of the HAE/HSL2-SERK complex.

RNA Sequencing of Col-0, hae-3 hsl2-3, and hae-3 hsl2-3 serk1-L326F

We next performed an RNA sequencing (RNA-Seq) experiment on floral receptacle-derived RNA to examine the transcriptome of the hae-3 hsl2-3 serk1-L326F mutant in relation to the parental hae-3 hsl2-3 mutant and the grandparental Col-0. We hypothesized that the serk1 suppressor would exhibit a reversion of gene expression levels from the hae-3 hsl2-3 parent toward the Col-0 grandparent (output of differential expression analysis in Supplemental Data Set S1).

First, we assessed transcript abundance measurements for the HAE/HSL2 marker genes QRT2 and PGAZAT (Fig. 3). These genes encode polygalacturonases involved in the breakdown of pectin in the middle lamella (Ogawa et al., 2009). The double qrt2 pgazat mutant exhibits a weak abscission delay, and expression of these genes is strongly reduced in the hae hsl2 mutant (Ogawa et al., 2009; Niederhuth et al., 2013b). Thus, we consider the expression of these genes useful markers for HAE/HSL2 pathway activity, although it should be noted that they are likely only two of many functionally relevant hydrolase genes regulated by HAE/HSL2. Consistent with the hypothesis that the hae-3 hsl2-3 serk1-L326F mutant is gain of function, the transcript abundance of QRT2 is increased to an approximately wild-type level (Fig. 3). In contrast, PGAZAT has a greater than 5-fold increase compared with the wild-type grandparent (Fig. 3). These results are consistent with a model where there is activation of a SERK1-regulated abscission signaling pathway in the hae-3 hsl2-3 serk1-L326F mutant.

Figure 3.

Figure 3.

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Transcript abundance measurements for abscission hydrolase genes PGAZAT and QRT2. A, Log2(fold change) relative to wild-type average fragments per kilobase per million reads for abscission hydrolase genes QRT2 and PGAZAT. n = 3 observations from tissue isolated from three independent pools of plants. Asterisks denote P < 0.05 at false discovery rate (FDR) of 0.05. B, Log2(fold change) relative to wild-type average fragments per kilobase per million reads for abscission hydrolase genes QRT2 and PGAZAT. n = 3 observations from tissue isolated from three independent pools of plants. Asterisks denote P < 0.05 at FDR of 0.05.

To detect global patterns in gene expression changes in the hae-3 hsl2-3 serk1-L326F mutant, we performed Gene Ontology (GO) analysis comparing all three genotypes (Supplemental Table S3). The most statistically significant findings are terms enriched in the Col-low/hae-3 hsl2-3 serk1-L326F-high and hae-3 hsl2-3-low/hae-3 hsl2-3 serk1-L326F-high comparisons and include genes associated with terms such as response to stimulus, response to stress, and response to biotic stimulus, among other terms associated with response to biotic stress (partial list in Table 1). Overall, these results suggest that there is a SERK1-mediated signaling pathway overactivated in hae-3 hsl2-3 serk1-L326F regulating both abscission and biotic stress response signaling.

Table 1. GO terms associated with differentially expressed genes in Col-low/hae-3 hsl2-3 serk1-L326F-high and Col-low/serk1-L326F-high comparisons.

Description FDR
Col-low/hae-3 hsl2-3 serk1-L326F-high
 Response to stimulus 1.2E-93
 Response to stress 1.8E-80
 Response to biotic stimulus 3.3E-65
 Multiorganism process 1.6E-57
 Response to external stimulus 2.3E-56
 Response to endogenous stimulus 6.3E-29
 Cell communication 1.1E-28
 Signal transduction 2.5E-27
 Cell death 2E-17
 Secondary metabolic process 3.9E-14
Col-low/serk1-L326F-high
 Response to stimulus 1E-51
 Response to stress 4.4E-37
 Response to biotic stimulus 1.6E-36
 Response to external stimulus 7.8E-33
 Multiorganism process 2.3E-32
 Response to endogenous stimulus 2.3E-14
 Response to abiotic stimulus 2.3E-14
 Secondary metabolic process 6E-11
 Cell communication 4.4E-08
 Catabolic process 5.4E-08
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As an additional control, we performed an RNA-Seq experiment comparing Col-0 with the single loss-of-function mutant serk1-1 and the single putative gain-of-function mutant serk1-L326F. In this experiment, we observed a moderate but statistically significant reduction in expression for QRT2 and PGAZAT in the loss-of-function serk1-1 mutant compared with the wild type (Fig. 3). The magnitude of this effect was less than that observed in the hae hsl2 double mutant, consistent with a model where SERK1 is one of a set of redundant SERK genes regulating abscission. In contrast, in the serk1-L326F single mutant, we observed a statistically significant increase in both QRT2 and PGAZAT expression compared with the wild type (Fig. 3). These results are consistent with a model where SERK1 positively regulates abscission signaling and the serk1-1 and serk1-L326F mutations are loss of function and gain of function, respectively.

In addition, in the Col-low/serk1-L326F-high and serk1-1-low/serk1-L326F-high comparisons, GO analysis identified a strong enrichment in terms such as response to stimulus, response to stress, and response to biotic stimulus, similar to the hae-3 hsl2-3 serk1-L326F mutant (Table 1; Supplemental Table S3). These results are consistent with a model serk1-L326F mutant as a gain-of-function allele broadly activating intracellular signaling. Additionally, we performed quantitative phenotyping of the wild type compared with the single serk1-1 and serk1-L326F mutants (Supplemental Fig. S2). We observed that wild-type abscission occurred at a median floral position between 4 and 5 (floral position 1 is defined as the first flower postanthesis, with each older flower increasing in position by 1; Supplemental Fig. S2). We observed the serk1-L326F mutant abscising slightly earlier than the wild type at median floral position 4, although this difference was not statistically significant (Supplemental Fig. S2). The single serk1-1 mutant, in contrast, abscised at a median position of 6, which is highly statistically significantly delayed compared with both the wild type and serk1-L326F (Supplemental Fig. S2). These results further support that there is a reduction in signaling in the loss-of-function serk1-1 mutant. They also suggest that, in serk1-L326F, enhanced signaling does not appear to dramatically alter the timing of abscission compared with the wild type. Interestingly, for both the serk1-L236F single mutant and the hae-3 hsl2-3 serk1-L326F suppressor mutant, we observed enlarged and disordered abscission zones following abscission by floral position 10, suggesting that signaling is not being properly regulated and/or attenuated (Supplemental Fig. S2).

The sum of these results indicates that these SERK1 suppressor mutations are hypermorphic and induce signaling downstream of the plasma membrane through a MAP kinase cascade. This signaling activates broad transcriptional reprogramming, including genes associated with cell wall modification during abscission, as well as biotic stress response signaling.

The Suppression Effect Does Not Require SERK1 Protein Kinase Activity

We next investigated potential biochemical mechanisms of hae hsl2 suppression by the SERK1 mutations. We hypothesized that the protein kinase domain mutations might overactivate the SERK1 protein kinase domain, causing constitutive phosphorylation of cellular substrates. Contrary to this hypothesis, in vitro autophosphorylation analysis did not identify a difference in the autophosphorylation level between wild-type SERK1 and the three protein kinase domain mutations with the strongest suppression effect (Fig. 4). This result suggests that these mutations do not alter intrinsic protein kinase activity of the SERK1 protein.

Figure 4.

Figure 4.

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Analysis of kinase activity on the hae-3 hsl2-3 suppression effect. A, In vitro autophosphorylation after 4 and 6 h of induction of recombinant MALTOSE BINDING PROTEIN (MBP)-SERK1 intracellular domain fusion proteins. n = 3 replicate cultures. Error bars represent se. WT, Wild type. B, Phenotypes of transgenic hae-3 hsl2-3 transformed with single and double mutant kinase-inactive, untagged variants of SERK1 (T1 generation).

We next hypothesized that the serk1 suppressor mutations alter a negative regulatory interaction in vivo that allows SERK1 to phosphorylate cellular substrates constitutively in a manner that does not alter SERK1 protein kinase activity. To test this hypothesis genetically, we used site-directed mutagenesis to create a SERK1pr::SERK1-L326F K330R transgene. This double mutant lacks an invariant catalytic Lys found in all protein kinases and lacks autophosphorylation activity in vitro (Supplemental Fig. S3). We hypothesized that this mutant would be unable to suppress the abscission-defective hae hsl2 phenotype due to abolition of its protein kinase activity. However, in the T1 generation, we found a spectrum of hae-3 hsl2-3 suppression phenotypes similar to the SERK1pr::SERK1-L326F single mutant T1 generation plants (∼45% strong or partial suppression in 22 lines; Fig. 4; Supplemental Table S2). We discovered that this effect also occurs with a version of SERK1-L326F K330R tagged with 2xHA (Supplemental Fig. S3). These results indicate that phosphorylation of a cellular substrate by the mutant kinase is not the cause of the suppression effect. We speculate that these mutations activate a SERK-guarding or -monitoring mechanism that senses some biochemical alteration of SERK proteins to transduce a signal downstream, independent of protein kinase activity.

Evidence That SOBIR1 Transduces Signaling Downstream of SERK1 in hae-3 hsl2-3 serk1-L326F

We next sought to understand downstream signaling mechanisms in the suppressor mutant. Recent work has shown that overexpression of a BAK1 transgene lacking the cytosolic kinase domain can activate pathogen response signaling via the RLK SOBIR1 (Domínguez-Ferreras et al., 2015). This result is reminiscent of our observation that signaling by serk1-L326F can induce intracellular signaling independent of the protein kinase activity of SERK1. In addition, SOBIR1 is specifically expressed in abscission zones and has been previously implicated in the regulation of floral abscission (Leslie et al., 2010). Thus, we hypothesized that SOBIR1 may function downstream of the suppressor mutations.

To test this model, we crossed the hae-3 hsl2-3 serk1-L326F mutant with the exonic SOBIR1 T-DNA mutant sobir1-12/evr-3. This mutant allele has previously been reported to suppress the abscission defect of nev-3 (Gao et al., 2009; Leslie et al., 2010). We hypothesized that SOBIR1 transduces signaling downstream of SERK1, and thus a sobir1 mutation would block the effect of the serk1-L326F mutation. In the F3 generation, we identified two quadruple homozygous individuals for the four mutations exhibiting strong abscission deficiency (Fig. 5). These results suggest that SOBIR1 transduces the signal downstream of the serk1-L326F mutation.

Figure 5.

Figure 5.

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Evidence that SOBIR1 functions downstream of SERK1 during abscission signaling. A, Phenotypes of hae-3 hsl2-3 serk1-L326F and hae-3 hsl2-3 serk1-L326F sobir1-12. B, Estimated log2(fold change) of transcript abundance measurements between hae-3 hsl2-3 serk1-L326F and hae-3 hsl2-3 serk1-L326F sobir1-12 for abscission hydrolase PGAZAT and pathogen response marker PR2. Asterisks denote P < 0.05. C, Transgenic complementation of the hae-3 hsl2-3 serk1-L326F sobir1-12 phenotype relies on kinase activity of SOBIR1 (T1 generation).

We performed quantitative PCR (qPCR) on the hae-3 hsl2-3 serk1-L326F sobir1-12 quadruple mutant and compared it with the hae-3 hsl2-3 serk1-L326F parent. We utilized a paired design where the difference of the normalized threshold counts for the two genotypes is calculated for each replicate to create a univariate relative gene expression measure. The null hypothesis is that there are no differences between the genotypes and the subtracted threshold counts will be centered around zero. The alternative hypothesis is that the differences will be centered around a nonzero value. We found that the abscission-associated hydrolase PGAZAT had, on average, an approximately log2(fold change) difference of 3, corresponding to an approximately 8-fold decrease of transcript abundance in the quadruple hae-3 hsl2-3 serk1-L326F sobir1 mutant (Fig. 5). We also tested the pathogen response marker PR2 and found that it exhibited an average log2(fold change) difference of 4, corresponding to an approximately 16-fold decrease in transcript abundance of PR2 in the quadruple mutant. Overall, these results confirm that there is a reduction in both abscission and pathogen signaling in the quadruple mutant (Fig. 5).

To confirm that the quadruple mutant phenotype is caused by the mutation in SOBIR1, we transformed the quadruple mutant with a transgene expressing either the wild-type SOBIR1 coding sequence fused to a FLAG tag or the same transgene with a mutation in the conserved catalytic Lys-377. We observed that the wild-type transgene complemented the abscission-deficient phenotype in a majority of T1 lines (58% of 36; Supplemental Table S2), whereas the K377R mutant did not complement any T1 lines examined (zero of 25; Fig. 5; Supplemental Table S2). We screened and found similarly expressing wild-type and K377R lines assayed with an anti-FLAG antibody (Fig. 5). Thus, SOBIR1 can transduce the abscission signal downstream of serk1-L326F in a protein kinase activity-dependent manner.

Evidence That BIR1 Negatively Regulates Abscission Signaling

Next, we sought to understand the mechanism by which the identified serk1 suppressor mutations might function through SOBIR1. The protein kinase BIR1 has been identified as an interactor of SERK proteins (Gao et al., 2009). In bir1 mutants, pathogen response signaling is constitutively activated, leading to extreme dwarfism and seedling lethality (Gao et al., 2009). This effect can be partially suppressed by mutation in SOBIR1 (Gao et al., 2009). Thus, BIR1 is thought to negatively regulate signaling by SOBIR1, possibly by acting in a guard complex. Based on these previously known genetic interactions, we hypothesized that BIR1 may negatively regulate signaling during abscission and that loss of function of BIR1 may lead to activation of SOBIR1 in abscission zones in a similar manner to that of serk1-L326F.

To test this hypothesis, we sought to combine the hae hsl2 double mutant with a loss-of-function bir1 mutant. We hypothesized that these plants would overactivate the SERK1-SOBIR1 abscission pathway and would therefore exhibit restored abscission and enhanced biotic stress response gene expression. bir1 null mutants are extremely dwarfed and typically die before flowering under standard growth conditions, rendering floral genetic studies difficult. As an alternative, artificial microRNA (amiRNA) targeting BIR1 has been shown to effectively mimic loss-of-function mutations in BIR1 (Gao et al., 2009). Therefore, we created two related BIR1 amiRNA constructs driven either by the HAE promoter alone or by a tandem 35S::HAEpr. We anticipated that the HAEpr would provide adequate expression levels in the abscission zone, while the tandem 35S::HAEpr would boost expression in abscission zones in the event that the HAEpr proved too weak to be effective.

In the T1, we found that two of 26 35S::HAEpr::amiRNA-BIR1 plants exhibited partial suppression of the hae-3 hsl2-3 abscission phenotype, as well as semidwarfism with yellowed leaves, while for HAEpr::amiRNA-BIR1, one of 16 plants exhibited similar partial abscission, yellow leaves, and semidwarfism (Fig. 6; Supplemental Table S2). Thus, loss of bir1 function appears to suppress the hae hsl2 abscission defect, phenocopying the gain-of-function mutation serk1-L326F. The leaf phenotype and semidwarfism are consistent with weak activation of autoimmunity. To examine these lines, we grew the T2 of one of the 35S::HAEpr lines and performed qPCR on RNA derived from the floral receptacle on BIR1 to examine the accumulation of transcript. Across three biological replicates, we observed a statistically significant log2(fold change) of 0.66 for BIR1 between hae-3 hsl2-3 and hae-3 hsl2-3 amiRNA-BIR1, corresponding to an approximately 40% reduction in BIR1 transcript accumulation (Fig. 6). We also tested transcript abundance of PGAZAT and PR2 and found an approximate log2(fold change) of five and 5.4 between hae-3 hsl2-3 and hae-3 hsl2-3 amiBIR1 for both genes, respectively, which corresponds to approximately 32- and 42-fold increases in expression of both genes in the amiRNA-BIR1 line (Fig. 6). These results are consistent with a model where BIR1 negatively regulates abscission signaling and that loss of function of BIR1 leads to high levels of abscission and biotic stress response gene expression, in a manner similar to the serk1-L326F mutations.

Figure 6.

Figure 6.

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Evidence that BIR1 negatively regulates abscission signaling. A, Phenotypes of hae-3 hsl2-3 and partially suppressed hae-3 hsl2-3 + HAEpr::amiRNA BIR1 (T1 generation). Bars = 1 cm. B, Estimated log2(fold change) of transcript abundance measurements between hae-3 hsl2-3 and hae-3 hsl2-3 amiBIR1 for BIR1, abscission hydrolase PGAZAT, and pathogen response marker PR2. Asterisks denote P < 0.05.

DISCUSSION

In this work, we have shown that a particular class of hypermorphic SERK1 mutations broadly activate intracellular signaling via the RLK SOBIR1. The phenotype of these hae hsl2 suppressor mutations in SERK1 can be phenocopied by loss of function of the negative regulator of signaling BIR1. Prior work has provided strong evidence that the function of BIR1 is to bind to SERK proteins and inhibit the activation of multiple pathways, including one regulated by SOBIR1. Taken together, these data suggest that BIR1 functions in abscission to inhibit overactivation of signaling by SOBIR1. These mutations should provide powerful insight into the activation of SOBIR1 and the regulation of this process by BIR1.

Prior work has shown that SOBIR1 is specifically expressed in floral abscission zones and that it is globally coexpressed with HAE (Leslie et al., 2010; Patharkar and Walker, 2015). This, combined with the genetic data presented in this study, suggests that SOBIR1 contributes to wild-type abscission signaling downstream of the SERK proteins. We hypothesize that the function of BIR1 during abscission is to moderate SERK-mediated activation of SOBIR1 to prevent overactivation of signaling. We further hypothesize that the serk1 suppressor mutations isolated in this screen interfere with the function of BIR1 by an unknown mechanism. Testing these hypotheses is a clear direction for future research. Notably, the single sobir1 mutant does not have an abscission defect (Leslie et al., 2010). This implies that there are parallel pathways regulating abscission downstream of the HAE/HSL2-SERK complex. An alternative explanation for these results is that, rather than act as a bona fide regulator of abscission signaling, SOBIR1 activation may instead be an undesired by-product of the activation of the SERK protein signaling complex, and this spurious activation of SOBIR1 may be promoted by the serk1 suppressor mutations identified in this screen. In this case, the function of BIR1 may simply be to prevent SERK protein-mediated activation of SOBIR1 altogether as a way to counteract this inadvertent signal. Given the specific expression pattern of SOBIR1 in floral abscission zones, this possibility seems unlikely, but it is one that cannot be ruled out until additional genetic evidence defining hypothetical parallel pathways including SOBIR1 comes to light.

This work also provides possible insight into the phenotype of the nev mutant. We have recently shown that the nev mutant exhibits widespread transcriptional reprogramming, including extensive induction of biotic stress response gene expression, and that this transcriptional reprogramming is partially reversed in the nev serk suppressor line (Taylor and Walker, 2018). Thus, it appears that there may be an aberrant signaling process regulated by SERK1 that is activated in nev that interferes with abscission zone function. This aberrant transcriptional reprogramming is reminiscent of that observed in the serk1-L326F mutant. Because loss-of-function mutations in both SERK1 and SOBIR1 can suppress the abscission defect of nev, this is evidence that the same pathway activated in the hae hsl2 serk1 suppressor mutants described in this article is activated in nev. Recent work has shown that the nev mutant exhibits overlignification of the abscission zone (Lee et al., 2018). This overlignification may be the result of aberrant signaling in nev and could plausibly interfere with normal cell wall modifications required for abscission. Thus, overlignification represents one possible physical explanation for the phenotype of the nev mutant. Why the activation of signaling would yield differing phenotypes in nev and the hae hsl2 serk1 suppressor mutants identified in this screen is an important question that will require additional research. It may be that cellular defects of the nev mutant render it more susceptible to abscission zone malfunction, or that aberrant signal intensity is higher in nev, or a combination of both. Much more work remains to understand nev and its suppressors as well as their relationship to the serk1 mutants identified in this screen, but this work will help guide future investigations in this area.

MATERIALS AND METHODS

Lines Used in This Study

Arabidopsis (Arabidopsis thaliana) mutants serk1-1 (SALK_044330C), serk2-1 (SALK_058020C), and sobir1-12 (SALK_ 050715) were obtained from the Arabidopsis Biological Resource Center (Alonso et al., 2003; Albrecht et al., 2005; Colcombet et al., 2005; Gao et al., 2009; Leslie et al., 2010). hae-3 hsl2-3, hae-3 hsl2-3 serk1-L326F, and hae-3 hsl2-3 serk1-L326F sobir1-12 have been deposited in the Arabidopsis Biological Resource Center. The hae-3 hsl2-3 serk1-L326F mutant was backcrossed to Col-0 five times. High-order mutant combinations were created by cross. All primers used in this study are listed in Supplemental Table S4.

Plant Growth Conditions

All plants with the exception of the Col-0/serk1-1/serk1-L326F RNA-Seq and abscission quantification experiment were grown in a 16-h light cycle, ∼125 μE m−2 s−1, at 22°C. Col-0/serk1-1/serk1-L326F RNA-Seq plants were grown in a 16-h light cycle, ∼60 μE m−2 s−1, 23°C. Col-0/serk1-1/serk1-L326F abscission quantification plants were grown in a 16-h light cycle, ∼150 μE m−2 s−1, 22°C. All plants were fertilized once at 3 weeks postgermination with 1× strength Miracle-Gro (Scotts Miracle-Gro).

Phenotyping

Qualitative suppression phenotypes were determined by lightly brushing the main inflorescence of young (less than 2 weeks post bolting) plants to remove remnant, abscised floral organs and classifying them according to highest similarity to hae-3 hsl2-3 (nonsuppressed), hae-3 hsl2-3 serk1-L326F/+ (partial suppression), and hae-3 hsl2-3 serk1-L326F (strong suppression). Phenotyping of transgenic lines was performed in the T1 generation.

Quantitative phenotyping of Col-0, serk1-1, and serk1-L326F was performed by utilizing our previously described petal-puller assay (Baer et al., 2016). In this assay, two paintbrushes are affixed at an angle to a rigid rod, with spacers present to create a consistent amount of force applied. We lightly dragged the inflorescence of plants at ∼2 weeks post bolting, then rotated the plants 90°, and performed the procedure again. The objective of this assay is to remove all remnant floral organs that have undergone abscission but are still loosely attached. Next, we counted the number of flowers that had nonabscised floral organs, starting from position 1. The first silique in which all floral organs had abscised was recorded. We performed this analysis for 10 plants of each genotype grown in the same flat. We performed a Wilcoxon rank-sum test on the floral positions to test for a shift in location of the median position of abscission.

RNA-Seq

RNA from six to 15 pooled stage 15 preabscission receptacles per replicate was isolated using the Trizol reagent (Life Technologies). The number of receptacles varied based on the number of stage 15 flowers for each genotype at the time of tissue harvesting. Each receptacle was dissected by taking a 1-mm section of floral tissue composed of 0.33 mm stem and 0.67 mm receptacle. Libraries were created using the TruSeq mRNA Library Prep Kit (Illumina). Libraries from each experiment were individually barcoded and run on a single lane of Illumina Sequencing. One replicate of Col-0, hae-3 hsl2-3, and hae-3 hsl2-3 serk1-L326F was run after the second backcross of the suppressor and hae-3 hsl2-3 mutants to Col-0 to obtain preliminary data, using the Illumina HiSeq 2500. The second and third replicates were performed on the fifth backcrosses and they, along with the Col-0/serk1-1/serk1-L326F experiment, were sequenced on an Illumina NextSeq 500.

Reads were mapped to TAIR 10 gene sequences and quantified using TopHat (v2.0.9) and Cufflinks (v2.1.1; Trapnell et al., 2012). We utilized default settings for alignments, and performed differential expression analyses using cuffdiff with default settings. Data were analyzed and visualized in R using ggplot2 (Trapnell et al., 2012). BAM files have been deposited at the Sequence Read Archive under BioProject accession number PRJNA430092.

The GO analyses were performed by outputting lists of genes from each comparison found to have a significant difference, with FDR set at 0.05. These lists were compared using agriGO (Du et al., 2010), using Fisher’s exact test, and Yekutieli FDR under dependency, with a 0.05 significance level, against the plant GO database.

hae-3 hsl2-3 Suppressor Screen

Two suppressor mutant screens were performed on a mutant derived from a cross of hae-3 hsl2-3 and a previously described erecta glabra (er gl) mutant, both in the Col-0 ecotype (Baer et al., 2016). hae-3 contains a missense mutation causing the amino acid residue Cys-222 to be substituted with Tyr in the extracellular domain, leading to degradation of the mutant protein by an endoplasmic reticulum-associated protein quality-control mechanism (Niederhuth et al., 2013a; Baer et al., 2016). hsl2-3 contains a missense mutation causing substitution of the amino acid residue Gly-360 with Arg in the HSL2 extracellular domain (Niederhuth et al., 2013a). The molecular defect of hsl2-3 is currently unknown. We isolated the hae-3 hsl2-3 er gl quadruple mutant, which fails to abscise and possesses the characteristic semidwarf phenotype of an er mutant and lacks trichomes. We used this mutant as a background for our screens to control for any wild-type seed contamination that could interfere with suppressor identification. Its short stature makes it convenient for dense planting to screen for floral phenotypes.

We performed an ethyl methanesulfonate suppressor screen by mutagenizing 50,000 hae-3 hsl2-3 er gl seeds and growing 50 pools of M1 seeds. Approximately 2,000 seeds from each M1 pool were grown in the M2 to screen for mutant phenotypes. We began initial characterization of four mutants that showed moderate to strong suppression. Two strong suppressors were selected for mapping by crossing to a hae hsl2 mutant in the Ler ecotype. In the F2, we selected strongly suppressed individuals, pooled the DNA, and sequenced on an Illumina HiSeq. Reads were aligned with Bowtie2 (version 2.2.6), and SNPs were analyzed with SAMtools (version 0.1.19) and the Bar Toronto mutant analysis pipeline to identify a linked region in both mutants on the long arm of chromosome 1 (Li et al., 2009; Austin et al., 2011; Langmead and Salzberg, 2012). Analysis of mutations revealed that each mutant had a distinct mutation in the SERK1 RLK gene. Since SERK1 is implicated in regulating abscission, it was our highest candidate (Lewis et al., 2010). The other strong mutant and an intermediate mutant were shown by linkage analysis in the F2 of a cross with Ler hae hsl2 to exhibit strong linkage to the marker NGA 111 near the SERK1 locus (Supplemental Fig. S1). Sanger sequencing of the coding regions of SERK1 in these lines revealed additional missense mutations.

Simultaneously, we performed an activation tagging screen in which 60,000 T1 and 180,000 T2 progeny were screened for suppression of the abscission-deficient phenotype of hae-3 hsl2-3 (Weigel et al., 2000). One line with weak suppression was shown, by thermal asymmetric interlaced-PCR and by backcross F2 segregation analysis, to have an unlinked T-DNA insertion upstream of At5g09880, a CC1-like splicing factor. However, analysis of the chromatogram of Sanger sequencing of pooled backcross F2 DNA showed a linked SNP in SERK1 in suppressed individuals, suggesting that a spontaneous mutation arose in the SERK1 gene during creation of this population (Supplemental Fig. S1).

Molecular Cloning

The SERK1pr::SERK1 construct was created by cloning an ∼5-kb fragment of the SERK1 locus including a stop codon into the pE2C entry vector using the NotI site. This was mutagenized by PCR to create SERK1pr::SERK1-L326F and SERK1pr::SERK1-L326F K330R. SERK1pr::SERK1 2xHA was created by mutagenizing SERK1pr::SERK1 by PCR-based site-directed mutagenesis to delete the stop codon and to create an in-frame fusion of SERK1 with 2xHA. This construct was mutagenized by PCR to create SERK1pr::SERK1-2xHA L326F, SERK1pr::SERK1-2xHA K330R, SERK1pr::SERK1-2xHA L326F K330R, and SERK1pr::SERK1-2xHA L326F K330E. All constructs were combined into the pGWB601 Gateway-compatible binary vector, transformed into Agrobacterium tumefaciens strain GV3101, transformed into Arabidopsis by floral dip, and selected with Basta (Clough and Bent, 1998; Nakamura et al., 2010).

Kinase inactive HAEpr::YDA-YFP was created by cloning the HAE promoter into pENTR-TOPO Kinase Inactive-YDA (Lampard et al., 2009). This construct was transferred to the binary vector pHGY by Gateway recombination, transformed into Arabidopsis by floral dip, and selected with hygromycin on Murashige and Skoog agar plates (Clough and Bent, 1998; Kubo et al., 2005).

MBP-SERK1 was created by PCR amplifying the intracellular domain of SERK1 from a cDNA library created from Arabidopsis flowers using SuperScript III reverse transcriptase (Thermo Fisher). This amplicon was cloned into the KpnI site of pMAL-cri and mutagenized by PCR to create pMAL-SERK1-KD-K330E, pMAL-SERK1-KD-L326F, pMAL-SERK1-KD-R330C, pMAL-SERK1-KD-R330H, and pMAL-SERK1-KD-L326F K330R.

The 35Spr::SOBIR1-FLAG construct was created by PCR amplification of the SOBIR1 gene from genomic DNA with the addition of a single C-terminal FLAG tag and stop codon. This fragment was cloned into pENTR/D-TOPO (Thermo Fisher). PCR-based mutagenesis was used to create 35Spr::SOBIR1-FLAG K377R. This construct was recombined into the Gateway-compatible pGWB602, which contains a 35S-driven promoter (Nakamura et al., 2010). Plants were transformed by floral dip and selected with Basta.

HAEpr::amiRNA BIR1 and 35S::HAEpr::amiRNA BIR1 constructs were created by cloning approximately 2 kb upstream of the HAE gene into the BamHI/NotI sites of the Gateway entry vector pE6c (Dubin et al., 2008). A PacI site was engineered downstream of HAEpr. The amiRNA BIR1 fragment was created by PCR from primers designed from WMD3 (http://wmd3.weigelworld.org). This fragment had a PacI site engineered on the 5′ end. This fragment was PacI digested and cloned into PacI/EcoRV-digested pE6c-HAEpr vector. This Gateway entry vector was recombined with pGWB601 (HAEpr::amiRNA BIR1) or pGWB602 (35S::HAEpr::amiRNA BIR1; Nakamura et al., 2010). These constructs were transformed into A. tumefaciens strain GV3101 and transformed into Arabidopsis by floral dip.

To control for cross-contamination, a minimum of one representative T1 individual from each SERK1 transgenic population was verified by using hae-3 hsl2-3 derived cleaved-amplified polymorphic sequence (dCAPS) markers (Supplemental Table S4) and by sequencing the SERK1 transgene across the mutation site(s) by analysis of a PCR product using SERK1 forward primer 5′-TGG​AAC​AAC​TGT​TAA​TGA​AAA​TCA​A-3′ and pE2c reverse primer 5′-AGT​CGG​GCA​CGT​CGT​AGG-3′.

A PCR product from a representative T1 individual from each HAEpr::YDA-YFP K429R population was subjected to Sanger sequencing using a HAE promoter-specific primer (5′-AGG​CAG​AGT​GCT​TGT​GGA​GAC​G-3′) and a YDA-specific primer (5′-CAG​GTG​CCA​TCC​AAT​ATG​GGC​TC-3′). The hae-3 hsl2-3 serk1-L326F T1 individual also was subjected to genotyping with serk1-L326F dCAPS primers (Supplemental Table S4).

The strongly expressing hae-3 hsl2-3 serk1-L326F sobir1-12 transgenic individuals were validated by genotyping with hae-3, hsl2-3, and serk1-L326F dCAPS markers as well as SOBIR1 wild-type and T-DNA-specific primers.

A single T2 hae-3 hsl2-3 + 35S::HAEpr::amiRNA BIR1 plant was validated by hae-3 and hsl2-3 dCAPS markers and by PCR amplifying and Sanger sequencing across the amiRNA sequence using HAEpr primer 5′-TTC​ACA​TGG​ATG​TAT​ACT​ATT​GCC​TCC​T-3′ and amiRNA primer B 5′-GCG​GAT​AAC​AAT​TTC​ACA​CAG​GAA​ACA​G-3′ to ensure that it correctly aligned to the output generated by WMD3.

All constructs were created using PFU Ultra II High-Fidelity polymerase and Sanger sequence verified across the entire coding region (Agilent). All primers are listed in Supplemental Table S4.

Immunoblotting

Three whole flowers, starting with a stage 15 flower and proceeding with the next two oldest flowers, were frozen and ground in microcentrifuge tubes, resuspended in 30 µL of SDS sample buffer, and boiled for ∼3 min. Ten microliters of each sample was run on an 8% (w/v) acrylamide gel, blotted to a nitrocellulose membrane, stained with Ponceau S, and imaged. Blots were blocked with 4% (w/v) BSA in phosphate-buffered saline with 0.1% Tween 20 (PBS-T) for 1 h, probed with anti-HA-horseradish peroxidase (HRP) or anti-FLAG antibody at 1:1,000 dilution for 1 h at room temperature or overnight at 4°C, respectively, and then rinsed with PBS-T four times for 5 min each (anti-HA-HRP antibody [Roche clone 3F10] and anti-FLAG antibody [Sigma M2]). HA blots were directly imaged. FLAG blots were incubated with anti-mouse HRP (1:2,500 dilution in 1% BSA in PBS-T; Cell Signaling Technologies) for 1 h at room temperature and rinsed with PBS-T four times for 5 min each. Blots were imaged by incubation with a chemiluminescent substrate (SuperSignal West Pico; Life Technologies) and imaged using a Bio-Rad ChemiDoc.

qPCR

RNA from five stage 15 receptacles was isolated using the Trizol reagent, from which cDNA was synthesized following DNase I treatment. Three or five biological replicates per reaction were performed. Tissue was harvested in balanced batches one replicate at a time to facilitate the use of a paired Student’s t test. We used ABsolute SYBR Green Master Mix (2×; Thermo Fisher) and a Bio-Rad CFX96 thermal cycler to estimate threshold counts during the log-linear phase of amplification. Data analysis was performed in Microsoft Excel. We utilized reference gene AT5G25760 for normalization for the hae-3 hsl2-3 serk1-L326F sobir1-12 experiment and the geometric mean of AT3G01150 and AT2G28390 for BIR1 experiments (Czechowski et al., 2005). An eightfold serial dilution was used to calculate PCR primer efficiency and determine estimated fold change levels.

In Vitro Autophosphorylation

In vitro autophosphorylation assays were carried out exactly as described (Taylor et al., 2013). In brief, expression of MBP-SERK1 was induced by adding isopropyl-β-d-thiogalactopyranoside to rapidly growing Escherichia coli to a final concentration of 0.1 mm. These cultures were then allowed to grow for 4 or 6 h, after which 100 µL of liquid culture was spun down, resuspended in 100 µL of 1× SDS sample buffer, and boiled for 3 min. Ten microliters of the whole-cell lysate was then run on an 8% (w/v) acrylamide gel, after which the gel was fixed by incubation in 50% (v/v) ethanol/10% (v/v) acetic acid overnight. The gel was rehydrated by soaking in distilled, deionized water two times for 30 min and then immersed in one-third-strength Pro-Q Diamond dye for 2 h in the dark on a rotating platform (Thermo Fisher). The gel was destained in 20% (v/v) acetonitrile and 50 mm sodium acetate (pH 4) four times for 30 min each time. Then, it was soaked in distilled, deionized water twice for 10 min before imaging on a Bio-Rad ChemiDoc using the default Pro-Q Diamond settings. Finally, the gel was stained with Coomassie Blue and imaged using a Bio-Rad GelDoc. Band quantity estimates were performed using the built-in Bio-Rad software.

Statistical Analysis

For testing the timing of abscission in serk1-1, serk1-L326F, and the wild type, a Wilcoxon rank-sum test was performed on the positions of abscission using a Bonferroni correction for multiple testing. For all qPCR experiments, two genotypes were compared by performing a paired Student’s t test on the delta-delta-Ct between the gene of interest and previously published reference gene(s) under the null hypothesis that the average of the differences was equal to 0 (Dussault and Pouliot, 2006; Yuan et al., 2006).

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: HAE, NM_118991; HSL2, NM_125968.3; SERK1, NM_105841.4; SOBIR1, NM_128746.3; and BIR1, NM_124213.5.

Supplemental Data

The following supplemental materials are available.

Acknowledgments

We thank the skilled staff at the University of Missouri DNA Core Facility for Illumina Sequencing services. We also thank Melody Kroll (University of Missouri) for thoughtful review of the article. We thank Tsuyoshi Nakagawa (Shimane University) for providing the pGWB601/pGWB602 binary vectors containing the bar gene, which was identified by Meiji Seika Kaisha. The Gateway entry vector pE2C was obtained from Addgene. bak1-5 was kindly provided by Antje Heese (University of Missouri). The YDA-KI plasmid was kindly provided by Dominique Bergmann (Stanford University).

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