Genetic and phenotypic characterization of a novel brachytic2 allele of maize

The maize brachytic2 (br2) gene encodes a protein homologous to the Arabidopsis ABCB1 protein, which actively transports chemically diverse substrates across cellular membranes. This includes indole-3-acetic acid (IAA), the most abundant natural form of auxin, a key regulator of plant growth and tropisms. Defects in auxin transport are responsible for the short stature of br2 mutants. We describe a novel br2 mutant allele present in NC238, an inbred maize of medium stature and demonstrate that the mutant allele br2-NC238 is caused by the insertion of a novel transposon in intron IV of the br2 gene. A tall revertant of NC238 (NC238-rev) appeared spontaneously as a result of excision of the transposon from the br2 gene. This event yielded two completely isogenic versions of the NC238 inbred, one brachytic (NC238-ref) and one tall (NC238-rev). The presence of the transposon in br2-NC238 alters normal splicing of the transcript, producing several splicing forms. One splicing form, ABCB1-T02, was abundantly expressed in NC238-ref, while its expression was very weak in tall NC238-rev plants. Differences between NC238-rev and NC238-ref were also detected in the expression pattern of auxin transporter genes. Morphological changes associated with the br2-NC238 mutant allele included reduced length and increased diameter of the lower internodes. The shortening of plant stature was confirmed in the B73 genetic background by introgression of the br2-NC238. Our results demonstrate that br2-NC238 reduces plant height without altering other morphological traits, and might thus be useful in maize breeding programs when reduction of stature is desired

Molecular and Functional Analyses of a Maize Autoactive NB-LRR Protein Identify Precise Structural Requirements for Activity

<div><p>Plant disease resistance is often mediated by nucleotide binding-leucine rich repeat (NLR) proteins which remain auto-inhibited until recognition of specific pathogen-derived molecules causes their activation, triggering a rapid, localized cell death called a hypersensitive response (HR). Three domains are recognized in one of the major classes of NLR proteins: a coiled-coil (CC), a nucleotide binding (NB-ARC) and a leucine rich repeat (LRR) domains. The maize NLR gene <i>Rp1-D21</i> derives from an intergenic recombination event between two NLR genes, <i>Rp1-D</i> and <i>Rp1-dp2</i> and confers an autoactive HR. We report systematic structural and functional analyses of Rp1 proteins in maize and <i>N</i>. <i>benthamiana</i> to characterize the molecular mechanism of NLR activation/auto-inhibition. We derive a model comprising the following three main features: Rp1 proteins appear to self-associate to become competent for activity. The CC domain is signaling-competent and is sufficient to induce HR. This can be suppressed by the NB-ARC domain through direct interaction. In autoactive proteins, the interaction of the LRR domain with the NB-ARC domain causes de-repression and thus disrupts the inhibition of HR. Further, we identify specific amino acids and combinations thereof that are important for the auto-inhibition/activity of Rp1 proteins. We also provide evidence for the function of MHD2, a previously uncharacterized, though widely conserved NLR motif. This work reports several novel insights into the precise structural requirement for NLR function and informs efforts towards utilizing these proteins for engineering disease resistance.</p></div

Investigating the functional domains of Rp1-D21 and Rp1-D to trigger a hypersensitive response (HR).

<p>(A) Schematic diagram of the Rp1-D21 and Rp1-D domain structure and the derived fragments used for agro-infiltration of <i>N</i>. <i>benthamiana</i>. The different domains are indicated using different colors: CC (purple); NB-ARC (red); LRR (dark blue). The positions of the amino acids that form the domain boundaries are indicated on the top and the abilities to induce (+) or not induce (-) HR are listed on the right of each construct. (B) The HR phenotype of the constructs fused with a C-terminal EGFP tag and transiently expressed in <i>N</i>. <i>benthamiana</i>. Representative leaves were photographed at 3 days post infiltration (dpi, left). Total protein was extracted from agro-infiltrated leaves at 30 hours post infiltration (hpi), and anti-GFP antibody was used to detect the expression of fused proteins. Equal loading of protein samples was shown by Ponceau-S staining of Rubisco subunit (right, below). The results shown were from different domains of Rp1-D21, and similar results were observed from different domains of Rp1-D. (C) The phenotype of the constructs fused with a C-terminal 3×HA tag and transiently expressed in <i>N</i>. <i>benthamiana</i>. A representative leaf was photographed at 3 dpi (left), and the same leaf was cleared by ethanol (middle). Total protein was extracted from agro-infiltrated leaves at 30 hpi, and anti-HA antibody was used to detect the expression of the fused proteins. Equal loading of protein samples was shown by Ponceau-S staining of Rubisco subunit (right). The results shown were from different domains of Rp1-D21, and similar results were observed from different domains of Rp1-D. The experiments were repeated three times with the same results.</p

Model of the intra-molecular interactions controlling auto-inhibition or auto-activation in Rp1 proteins.

<p>In the auto-inhibited state (left), the NB-ARC domain inhibits CC-induced HR via intra-molecular interaction involving the ARC2 domain. In the auto-activated state (right), the LRR suppresses an inhibitory effect of the NB-ARC on the CC through direct interaction with the NB-ARC which destabilizes the CC/NB-ARC interaction. Blue and pink triangles indicate ADP and ATP, respectively. Arrows indicate intra-molecular interactions. The positions of amino acids (D82, R125, N1184 and the C-terminal 16 amino acids) which are important for activity are labeled in red. The rationale behind the models is explained in the main text.</p

Sequence alignment of Rp1-D, Rp1-dp2 and Rp1-D21.

<p>The CC (coiled-coil), NB-ARC (nucleotide binding), ARC1 (APAF1, <i>R</i> gene products and CED-4), ARC2 and LRR (leucine-rich-repeat) domains were indicated by bars with cyan, green, dark blue, pink and red colors, respectively. The conserved motifs (EDVID, P-loop, GLPL, RNBS-D, MHD1 and MHD2) were indicated by orange bars and labeled below the sequences. The recombination point of Rp1-D21 was labeled by cyan bar. Patches 1 and 2 are the major difference regions between Rp1-D and Rp1-dp2 in the ARC2 domain. The positions of the mutations in the intragenic Rp1-D21 suppressor from maize were labeled by blue color. The landmark positions of recombination points in the constructs listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004674#ppat.1004674.g006" target="_blank">Fig. 6</a> were labeled by numbers with red color. The black and light blue shaded regions represent 100% and above 50% similarity of the amino acids, respectively.</p

Rp1-D21 triggers a hypersensitive response phenotype when transiently expressed in <i>N</i>. <i>benthamiana</i>.

<p>(A) Rp1-D21, Rp1-D and Rp1-dp2 proteins fused with a C-terminal EGFP tag were agro-infiltrated into <i>N</i>. <i>benthamiana</i>, with an empty vector (EV) as a negative control. A representative leaf was photographed at 3 days post infiltration (dpi, left). Total protein was extracted from agro-infiltrated leaves at 30 hours post infiltration (hpi), and anti-GFP antibody was used to detect the expression of the fused proteins (right). The sizes of the proteins were labeled on the right. Equal loading of protein samples was shown by Ponceau-S staining of the Rubisco subunit (below, right). (B) Rp1-D21, Rp1-D and Rp1-dp2 proteins fused with a C-terminal 3×HA tag were agro-infiltrated into <i>N</i>. <i>benthamiana</i>, with empty vector (EV) as a negative control. A representative leaf was photographed at 3 dpi (left). Total protein was extracted from agro-infiltrated leaves at 30 hpi, and anti-HA antibody was used to detect the expression of the fused proteins. Equal loading of protein samples was shown by Ponceau-S staining of Rubisco subunit (below, right). These experiments were repeated three times with the same results.</p

Functional characterization of the MHD2 motif and the P-loop motif.

<p>(A) Multiple sequence alignment of the MHD motifs from Rp1-D, Rp1-D21 and Rp1-dp2. Positions of the MHD1 and MHD2 motifs are indicated. The numbers indicated the positions of the aspartate (D) corresponding to Rp1-dp2. (B) The MHD1 and MHD2 point mutations in Rp1-dp2 background were generated and transiently expressed in <i>N</i>. <i>benthamiana</i>. (C) Investigating the MHD2 point mutations in V1 and V16, two recombinant constructs that were not autoactive as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004674#ppat.1004674.g006" target="_blank">Fig. 6</a>. (D) Point mutations in P-loop motif of Rp1-D21 and Rp1-dp2(D517V) caused the loss of the ability to induce HR. EV (empty vector) as a negative control. The picture of hypersensitive response (HR) phenotype was taken at 3 days post-infiltration and total protein was detected by anti-HA antibody from agro-infiltrated leaves at 30 hours post infiltration. Equal loading of protein samples was shown by Ponceau-S staining of Rubisco subunit. The experiments were repeated three times with the same results.</p

Schematic diagram of the intra-molecular interactions and HR phenotype of the constructs indicated, summarizing the data shown in S7 Fig. in which the various domains were co-expressed and co-immunoprecipitated <i>in trans</i>.

<p>The gene structure has been divided into CC, NB-ARC and LRR domains. The amino acid position of the recombination site of each construct was indicated above the construct. For each construct, the strength of the hypersensitive response (HR) resulting from transient expression of each full length molecule in <i>N</i>. <i>benthamiana</i> was scored on a 0 (no HR) to 5 (strong HR) scale. The ability to form or not to form intra-molecular interactions among different domains was indicated by arrows or arrows with crosses, respectively.</p