Identification of endogenous small peptides involved in rice immunity through transcriptomics- and proteomics-based screening

Small signalling peptides, generated from larger protein precursors, are important components to orchestrate various plant processes such as development and immune responses. However, small signalling peptides involved in plant immunity remain largely unknown. Here, we developed a pipeline using transcriptomics- and proteomics-based screening to identify putative precursors of small signalling peptides: small secreted proteins (SSPs) in rice, induced by rice blast fungus Magnaporthe oryzae and its elicitor, chitin. We identified 236 SSPs including members of two known small signalling peptide families, namely rapid alkalinization factors and phytosulfokines, as well as many other protein families that are known to be involved in immunity, such as proteinase inhibitors and pathogenesis-related protein families. We also isolated 52 unannotated SSPs and among them, we found one gene which we named immune response peptide (IRP) that appeared to encode the precursor of a small signalling peptide regulating rice immunity. In rice suspension cells, the expression of IRP was induced by bacterial peptidoglycan and fungal chitin. Overexpression of IRP enhanced the expression of a defence gene, PAL1 and induced the activation of the MAPKs in rice suspension cells. Moreover, the IRP protein level increased in suspension cell medium after chitin treatment. Collectively, we established a simple and efficient pipeline to discover SSP candidates that probably play important roles in rice immunity and identified 52 unannotated SSPs that may be useful for further elucidation of rice immunity. Our method can be applied to identify SSPs that are involved not only in immunity but also in other plant functions

Glycolysis regulates pollen tube polarity via Rho GTPase signaling

<div><p>As a universal energy generation pathway utilizing carbon metabolism, glycolysis plays an important housekeeping role in all organisms. Pollen tubes expand rapidly via a mechanism of polarized growth, known as tip growth, to deliver sperm for fertilization. Here, we report a novel and surprising role of glycolysis in the regulation of growth polarity in <i>Arabidopsis</i> pollen tubes via impingement of Rho GTPase-dependent signaling. We identified a <i>cytosolic phosphoglycerate kinase</i> (<i>pgkc-1</i>) mutant with accelerated pollen germination and compromised pollen tube growth polarity. <i>pgkc-1</i> mutation greatly diminished apical exocytic vesicular distribution of REN1 RopGAP (Rop GTPase activating protein), leading to ROP1 hyper-activation at the apical plasma membrane. Consequently, <i>pgkc-1</i> pollen tubes contained higher amounts of exocytic vesicles and actin microfilaments in the apical region, and showed reduced sensitivity to Brefeldin A and Latrunculin B, respectively. While inhibition of mitochondrial respiration could not explain the <i>pgkc-1</i> phenotype, the glycolytic activity is indeed required for PGKc function in pollen tubes. Moreover, the <i>pgkc-1</i> pollen tube phenotype was mimicked by the inhibition of another glycolytic enzyme. These findings highlight an unconventional regulatory function for a housekeeping metabolic pathway in the spatial control of a fundamental cellular process.</p></div

Effects of disrupting GAPDH on pollen tube polarity.

<p><b>(A)</b> Glycolytic pathway of GAPDH and PGK. <b>(B to G)</b> Pollen tube morphology of WT <b>(B)</b>, <i>pgkc-1</i> <b>(C)</b>, and <i>ren1-3</i> <b>(D)</b> plants subjected to mock treatment; pollen tube morphology of WT <b>(E)</b>, <i>pgkc-1</i> <b>(F)</b>, and <i>ren1-3</i> <b>(G)</b> plants treated with 40 μM CGP 3466B. Both <i>pgkc-1</i> and <i>ren1-3</i> plants were dramatically depolarized by CGP medium. Scale bar = 50 μm. <b>(H and I)</b> Pollen tube length <b>(H)</b> and width <b>(I)</b> of WT, <i>pgkc-1</i>, and <i>ren1-3</i> pollen tubes subjected to mock and 40 μM CGP treatment. Bars represent mean ± SEM. Asterisks indicate significant differences versus either single mutant as determined using Student’s <i>t</i>-test with either single mutant (** = p < 0.001). <b>(J)-(M)</b> Average signal intensity along WT pollen tubes subjected to mock or CGP treatment of <b>(J)</b> GFP- REN1, <b>(K)</b> CRIB4-GFP, <b>(L)</b> Lifeact-mEGFP, <b>(M)</b> EYFP-RABA4D. Measurements were performed as described in Materials and Methods. Fifteen pollen tubes from each sample were measured. The 0 μm label indicates the position of the apical tip.</p

F-actin dynamics in the <i>pgkc-1</i> mutant.

<p><b>(A)</b> Lifeact-mEGFP signal in WT and <i>pgkc-1</i> pollen tubes with mock or 1.5 nM LatB treatment. Scale bar = 5 μm. <b>(B)</b> Average GFP signal intensity along WT and <i>pgkc-1</i> pollen tubes with mock or 1.5 nM LatB treatment. Measurements were performed as described in Materials and Methods. Thirty-five pollen tubes were measured for each sample. The 0 μm indicates the position of the extreme tip. Orange line indicates WT pollen tube; red line indicates WT pollen tube treated with 1.5 nM LatB; gray line indicates <i>pgkc-1</i> pollen tube; black line indicates <i>pgkc-1</i> pollen tube treated with 1.5 nM LatB. Error bars on curves indicate standard error of the mean. <b>(C and D)</b> WT and <i>pgkc-1</i> pollen tube growth when subjected to mock medium <b>(C)</b> or 1.5 nM LatB <b>(D)</b> treatment. Scale bar = 50 μm. <b>(E to G)</b> WT and <i>pgkc-1</i> plant pollen germination <b>(E)</b>, pollen tube length <b>(F)</b>, and pollen tube width <b>(G)</b> when subjected to mock or 1.5 nM LatB treatment. Bars represent mean ± SEM. Asterisks indicate significant differences versus mock treatment as determined using Student’s <i>t</i>-test (** = p < 0.001).</p

Vesicle trafficking in the <i>pgkc-1</i> mutant.

<p><b>(A)</b> EYFP-RABA4D signal in WT and <i>pgkc-1</i> pollen tubes subjected to mock or 0.4 μM BFA treatment. Scale bar = 5 μm. <b>(B)</b> EYFP-RABA4D signal intensity. Measurements were performed as described in Materials and Methods. Fifteen to twenty pollen tubes from each sample were measured. 0 μm indicates the position of apical tip. Error bars on curves indicate standard error. <b>(C and D)</b> WT and <i>pgkc-1</i> pollen tube morphology when subjected to <b>(C)</b> mock and <b>(D)</b> 0.4 μM BFA treatment. Scale bar = 50 μm. <b>(E to G)</b> WT and <i>pgkc-1</i> plant pollen germination <b>(E)</b>, pollen tube length <b>(F)</b>, and pollen tube width <b>(G)</b> when subjected to mock or 0.4 μM BFA treatment. Bars represent mean ± SEM. Asterisks indicate significant differences versus mock treatment as determined using Student’s <i>t</i>-test (** = p < 0.001).</p

<i>pgkc-1</i> mutant exhibits enhanced pollen germination and growth depolarization.

<p><b>(A)</b> WT pollen tube morphology. <b>(B)</b> <i>pgkc-1</i> (SALK_066422C) pollen tube morphology. <b>(C)</b> Complemented <i>pgkc-1</i> pollen tube morphology. Scale bar = 50 μm. <b>(D)</b> Pollen germination rate at 3 h and 9 h, respectively. <i>pgkc-1</i> germinated at higher rates than WT and complemented pollen, especially at the early time point. (<b>E</b>) Pollen tube length of WT, <i>pgkc-1</i> mutant, and genetically complemented <i>pgkc-1</i> plants at 9 h after germination. <b>(F)</b> Pollen tube width of WT, <i>pgkc-1</i> mutant, and genetically complemented <i>pgkc-1</i> plants at 9 h after germination. Bars represent mean ± SEM. Asterisks indicate significant differences (** = p < 0.001) versus WT as determined by Student’s <i>t</i>-test.</p

ROP1 signaling in the <i>pgkc-1</i> mutant.

<p><b>(A)</b> Active ROP1 visualized by CRIB4-GFP signal in WT and <i>pgkc-1</i> pollen tubes. Scale bar = 5μm. <b>(B)</b> Average CRIB4-GFP signal intensity along WT and <i>pgkc-1</i> pollen tubes. <b>(C)</b> GFP-REN1 localization in WT and <i>pgkc-1</i> pollen tubes. Scale bar = 5 μm. <b>(D)</b> Average GFP-REN1 signal intensity along WT and <i>pgkc-1</i> pollen tubes. Measurements were performed as described in Materials and Methods. Fifteen pollen tubes from each sample were measured. The 0 μm label indicates the position of the apical tip. Error bars on curves indicate standard error of the mean. <b>(E to G)</b> Pollen tube morphology of <i>pgkc-1</i> <b>(E)</b>, <i>ren1-3</i> <b>(F)</b>, and <i>ren1-3/pgkc-1</i> double mutant plants <b>(G)</b>. Scale bar = 50 μm. <b>(H and I)</b> Pollen tube length <b>(H)</b> and width <b>I)</b> of WT, <i>pgkc-1</i>, <i>ren1-3</i>, and <i>ren1-3/pgkc-1</i> double mutant plants. Bars represent mean ± SEM. Asterisks indicate significant differences versus single mutant plant as determined using Student’s <i>t</i>-test (** = p < 0.001).</p

<i>gapcp1/gapcp2</i> double mutant is also defective in pollen tube polarity.

<p><b>(A)</b> WT pollen tubes. <b>(B)</b> <i>gapcp1/gapcp2</i> double mutant pollen tubes. Scale bar = 50μm. <b>(C)</b> Average length of pollen tubes. <b>(D)</b> Average width of pollen tubes. Bars represent mean ± SEM. Asterisks indicate significant differences versus WT as determined using Student’s <i>t</i>-test (** = p < 0.001).</p