Data_Sheet_1_GhSNAP33, a t-SNARE Protein From Gossypium hirsutum, Mediates Resistance to Verticillium dahliae Infection and Tolerance to Drought Stress.DOCX

<p>Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins mediate membrane fusion and deliver cargo to specific cellular locations through vesicle trafficking. Synaptosome-associated protein of 25 kDa (SNAP25) is a target membrane SNARE that drives exocytosis by fusing plasma and vesicular membranes. In this study, we isolated GhSNAP33, a gene from cotton (Gossypium hirsutum), encoding a SNAP25-type protein containing glutamine (Q)b- and Qc-SNARE motifs connected by a linker. GhSNAP33 expression was induced by H₂O₂, salicylic acid, abscisic acid, and polyethylene glycol 6000 treatment and Verticillium dahliae inoculation. Ectopic expression of GhSNAP33 enhanced the tolerance of yeast cells to oxidative and osmotic stresses. Virus-induced gene silencing of GhSNAP33 induced spontaneous cell death and reactive oxygen species accumulation in true leaves at a later stage of cotton development. GhSNAP33-deficient cotton was susceptible to V. dahliae infection, which resulted in severe wilt on leaves, an elevated disease index, enhanced vascular browning and thylose accumulation. Conversely, Arabidopsis plants overexpressing GhSNAP33 showed significant resistance to V. dahliae, with reduced disease index and fungal biomass and elevated expression of PR1 and PR5. Leaves from GhSNAP33-transgenic plants showed increased callose deposition and reduced mycelia growth. Moreover, GhSNAP33 overexpression enhanced drought tolerance in Arabidopsis, accompanied with reduced water loss rate and enhanced expression of DERB2A and RD29A during dehydration. Thus, GhSNAP33 positively mediates plant defense against stress conditions and V. dahliae infection, rendering it a candidate for the generation of stress-resistant engineered cotton.</p

Molecular and Functional Characterization of a Polygalacturonase-Inhibiting Protein from <i>Cynanchum komarovii</i> That Confers Fungal Resistance in Arabidopsis

<div><p>Compliance with ethical standards: This study did not involve human participants and animals, and the plant of interest is not an endangered species.</p><p>Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat proteins that plants produce against polygalacturonase, a key virulence agent in pathogens. In this paper, we cloned and purified CkPGIP1, a gene product from <i>Cynanchum komarovii</i> that effectively inhibits polygalacturonases from <i>Botrytis cinerea</i> and <i>Rhizoctonia solani</i>. We found the expression of <i>CkPGIP1</i> to be induced in response to salicylic acid, wounding, and infection with <i>B</i>. <i>cinerea</i> and <i>R</i>. <i>solani</i>. In addition, transgenic overexpression in Arabidopsis enhanced resistance against <i>B</i>. <i>cinerea</i>. Furthermore, CkPGIP1 obtained from transgenic Arabidopsis inhibited the activity of <i>B</i>. <i>cinerea</i> and <i>R</i>. <i>solani</i> polygalacturonases by 62.7–66.4% and 56.5–60.2%, respectively. Docking studies indicated that the protein interacts strongly with the B1-sheet at the N-terminus of the <i>B</i>. <i>cinerea</i> polygalacturonase, and with the C-terminus of the polygalacturonase from <i>R</i>. <i>solani</i>. This study highlights the significance of CkPGIP1 in plant disease resistance, and its possible application to manage fungal pathogens.</p></div

Purification and characterization of recombinant CkPGIP1.

<p><b>A.</b> Lane 1, total protein extract from <i>E</i>. <i>coli</i> expressing recombinant CkPGIP1; Lane 2, flow through from a Ni-IDA superflow column; Lane 3, purified CkPGIP1, with fusion tags removed. <b>B-C.</b> Agarose diffusion assay of <i>Botrytis cinerea</i> (<b>B</b>) and <i>Rhizoctonia solani</i> polygalacturonase (<b>C</b>) in the presence or absence of purified recombinant CkPGIP1. a, 25 μL enzyme; b, 25 μL enzyme + 15 μg CkPGIP1; c, 25 μL enzyme + 25 μL phosphate-buffered saline; d, 25 μL enzyme + 15 μg heat-denatured CkPGIP1. Inhibitory activity is inversely proportional to the size of the ring, and the present of inhibition is indicated by a smaller ring size between comparisons.</p

Inhibitory activity of crude protein extracts from wild type and transgenic Arabidopsis.

<p>Inhibitory activity was measured against <i>Botrytis cinerea</i> (<b>A</b>) and <i>Rhizoctonia solani</i> polygalacturonase (<b>B</b>). a-c, 15 μL crude enzyme + 15 μg crude protein extract from wild type and transgenic Arabidopsis lines 9 and 14; d-f, 15 μL crude enzyme + 15 μg heat-denatured crude protein extract from wild type and transgenic Arabidopsis lines 9 and 14; g, crude PGs. <b>C.</b> Inhibition rate of crude protein extracts from wild type and transgenic Arabidopsis. Error bars indicate standard error (n = 3). **, <i>P</i> < 0.01 by least significant difference against wild type.</p

The PGIP-specific consensus sequence xxLxLxx.NxLx..GxIPxxLxxL.xxL in leucine-rich repeat units of CkPGIP1.

<p>Secondary structure elements (sheets B1, B2, and 3₁₀-helix) are indicated for a homology model of CkPGIP1, which is based on PvPGIP2 (1OGQ) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146959#pone.0146959.ref026" target="_blank">26</a>]. Putative glycosylation sites are doubly underlined, while conserved C residues are marked using diamonds.</p

Phylogenetic analysis of CkPGIP1 and other known PGIPs.

<p>Amino acid sequences of CkPGIP1 (red box) and other known PGIPs were obtained from GenBank. The neighbor-joining tree was built in MEGA 5.1 based on a multiple sequence alignment.</p

Analysis of <i>CkPGIP1</i> expression by qPCR.

<p><b>A.</b><i>CkPGIP1</i> expression 0, 1, 6, 12, 24 and 48 h after induction with salicylic acid. <b>B-C.</b><i>CkPGIP1</i> expression 0, 1, 6, 24, 48 and 72 h after inoculation with <i>Botrytis cinerea</i> (<b>B</b>) and <i>Rhizoctonia solani</i> (<b>C</b>). <b>D.</b><i>CkPGIP1</i> expression 0, 1, 6, 12, 24 and 48 h after wounding. Expression was compared to control plants, which were treated with double distilled water. Data were collected from three independent biological replicates. The data are means± standard errors (n = 3). Asterisks indicate a significant difference compared with control [least significance differences (LSD), *P < 0.05].</p

Transgenic expression of CkPGIP1 increases resistance to <i>Botrytis cinerea</i> and <i>Rhizoctonia solani</i> in Arabidopsis.

<p><b>A-B.</b> Disease symptoms (top panel) and lesions (bottom panel) 6 d after wild type and transgenic Arabidopsis were inoculated with <i>B</i>. <i>cinerea</i> (<b>A</b>), and 5 d after inoculation with <i>R</i>. <i>solani</i> (<b>B</b>). Error bars are standard error (n = 3). *, <i>P</i> < 0.05 and **, <i>P</i> < 0.01 by least significant difference against wild type. <b>C</b>-<b>D.</b> Trypan blue staining of wild type <b>(C)</b> and transgenic Arabidopsis leaves <b>(D)</b> 4 d after inoculation with <i>B</i>. <i>cinerea</i>. <b>E-F.</b> Zoomed-in view of disease symptoms boxed in red in <b>(C)</b> and <b>(D)</b>. <b>G</b>-<b>J.</b> Trypan blue staining of wild-type <b>(G)</b> and transgenic Arabidopsis leaves <b>(H)</b> at 3 d post-inoculation with <i>R</i>. <i>solani</i>. (<b>H</b>) and (<b>I</b>) are enlarged views of areas boxed in red in (<b>G</b>) and (<b>H</b>).</p

Docking studies of CkPGIP1 with polygalacturonase from <i>Botrytis cinerea</i> and <i>Rhizoctonia solani</i>.

<p><b>A-B.</b> A concave site in CkPGIP1 interacts with the B1-sheet at the N-terminus of <i>B</i>. <i>cinerea</i> polygalacturonase (<b>A</b>), and with the C-terminus of <i>R</i>. <i>solani</i> polygalacturonase (<b>B</b>). When in complex with CkPGIP1, the substrate-binding site in the <i>B</i>. <i>cinerea</i> enzyme appears less exposed than in the enzyme from <i>R</i>. <i>solani</i> (circled in white).</p