Reference Genes in the Pathosystem Phakopsora pachyrhizi/ Soybean Suitable for Normalization in Transcript Profiling

Phakopsora pachyrhizi is a devastating pathogen on soybean, endangering soybean production worldwide. Use of Host Induced Gene Silencing (HIGS) and the study of effector proteins could provide novel strategies for pathogen control. For both approaches quantification of transcript abundance by RT-qPCR is essential. Suitable stable reference genes for normalization are indispensable to obtain accurate RT-qPCR results. According to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines and using algorithms geNorm and NormFinder we tested candidate reference genes from P. pachyrhizi and Glycine max for their suitability in normalization of transcript levels throughout the infection process. For P. pachyrhizi we recommend a combination of CytB and PDK or GAPDH for in planta experiments. Gene expression during in vitro stages and over the whole infection process was found to be highly unstable. Here, RPS14 and UbcE2 are ranked best by geNorm and NormFinder. Alternatively CytB that has the smallest Cq range (Cq: quantification cycle) could be used. We recommend specification of gene expression relative to the germ tube stage rather than to the resting urediospore stage. For studies omitting the resting spore and the appressorium stages a combination of Elf3 and RPS9, or PKD and GAPDH should be used. For normalization of soybean genes during rust infection Ukn2 and cons7 are recommended

A Small Cysteine-Rich Protein from the Asian Soybean Rust Fungus, Phakopsora pachyrhizi, Suppresses Plant Immunity

The Asian soybean rust fungus, Phakopsora pachyrhizi, is an obligate biotrophic pathogen causing severe soybean disease epidemics. Molecular mechanisms by which P. pachyrhizi and other rust fungi interact with their host plants are poorly understood. The genomes of all rust fungi encode many small, secreted cysteine-rich proteins (SSCRP). While these proteins are thought to function within the host, their roles are completely unknown. Here, we present the characterization of P. pachyrhizi effector candidate 23 (PpEC23), a SSCRP that we show to suppress plant immunity. Furthermore, we show that PpEC23 interacts with soybean transcription factor GmSPL12l and that soybean plants in which GmSPL12l is silenced have constitutively active immunity, thereby identifying GmSPL12l as a negative regulator of soybean defenses. Collectively, our data present evidence for a virulence function of a rust SSCRP and suggest that PpEC23 is able to suppress soybean immune responses and physically interact with soybean transcription factor GmSPL12l, a negative immune regulator.This article is published as Qi, Mingsheng, Tobias I. Link, Manuel Müller, Daniela Hirschburger, Ramesh N. Pudake, Kerry F. Pedley, Edward Braun, Ralf T. Voegele, Thomas J. Baum, and Steven A. Whitham. "A small cysteine-rich protein from the Asian soybean rust fungus, Phakopsora pachyrhizi, suppresses plant immunity." PLoS pathogens 12, no. 9 (2016): e1005827, doi: 10.1371/journal.ppat.1005827.</p

ニッケル製錬に関する研究． 特に本邦夏梅鉱に就て

種別: 卒業論

<i>Pp</i>EC23 interacts with soybean transcription factor <i>Gm</i>SPL12l.

<p>A. <i>Pp</i>EC23 and <i>Gm</i>SPL12l interaction confirmed by Y2H. The empty vector, pGADT7, was included as a negative control. SD/-LWHA and SD/-LW represents SD (-Leu/-Trp/-His/-Ade) and SD (-Leu/-Trp), respectively. The structural diagrams of <i>Pp</i>EC23_ns or truncated constructs of <i>Pp</i>EC23 are shown next to the corresponding strain. B. <i>Pp</i>EC23 and <i>Gm</i>SPL12l interaction detected in <i>N</i>. <i>benthamiana</i> nuclei by BiFC assay. YFP/GFP, yellow or green fluorescent protein epifluorescence. BF, bright field. DAPI signal was used as a nuclear marker. Arrows indicate nuclei. Representative images are shown (n ≥ 20). Bar = 20 μm. C. <i>Pp</i>EC23 and <i>Gm</i>SPL12l interaction confirmed by co-immunoprecipitation assay (CoIP). MW, molecular weight marker. The solid triangle indicates the band corresponding to the GFP-<i>Gm</i>SPL12l fusion protein. The white triangle indicates the GFP protein band. @FLAG and @GFP indicate detection using anti-FLAG and anti-GFP antibodies, respectively.</p

<i>Gm</i>SPL12l is a negative regulator of plant immunity.

<p>A. Phenotypes of soybean plants 21 dpi with BPMV. A. Soybean plants infected with the BPMV empty vector (BPMV:00) (left) and BPMV:<i>GmSPL12li</i> (right). Bar = 5 cm. Representative images are shown (n ≥ 6). B. Heights of plants at 21 dpi with BPMV:00 (white bars) and BPMV:<i>GmSPL12li</i> (black bars). A <i>t</i>-test was performed for the pair-wise comparison. The * indicates significant difference (<i>P</i> < 0.05). Six biological replicates were performed. C. Fold change of <i>GmSPL12l</i> (left panel) and <i>GmPR1a</i> (right panel) mRNA in BPMV:00 (white bars) and BPMV:<i>GmSPL12li</i> (black bars) plants. <i>GmAct1</i> was used as the internal reference gene. A <i>t</i>-test was performed for each comparison. The * indicates significant difference (<i>P</i> < 0.05). Four biological and four technical replicates were performed. D. Representative leaves from BPMV:00 (left) and BPMV:<i>GmSPL12li</i> (right) plants (n ≥ 18). Bar = 1 cm. E. Downy mildew symptoms on leaves of BPMV:00 (left) and BPMV:<i>GmSPL12li</i> (right) plants inoculated with <i>P</i>. <i>manshurica</i>. Leaves were photographed at 28 days post BPMV inoculation (7 days post <i>P</i>. <i>manshurica</i> inoculation). Representative third trifoliate leaves are shown (n ≥ 12). The assay was repeated three times. Bar = 1 cm.</p

SNPs of <i>PpEC23</i> genes among <i>P</i>. <i>pachyrhizi</i> isolates.

<p>SNPs of <i>PpEC23</i> genes among <i>P</i>. <i>pachyrhizi</i> isolates.</p

Strains and plasmids.

<p>Strains and plasmids.</p

<i>Pp</i>EC23 suppresses basal defense responses.

<p>A. Callose deposition in leaves of <i>A</i>. <i>thaliana</i> Col-0 induced by <i>Pst</i> DC3000, CUCPB5115 (ΔCEL)/EV, ΔCEL/<i>Pp</i>EC23_ns or ΔCEL/truncated constructs stained with aniline blue. The average number of callose spots ± standard deviation is listed under each representative image. Pair-wise <i>t</i>-tests were performed and a, b, c were designated groups with statistically significant difference. Bar = 50 μm. Representative images are shown (n ≥ 24). B. Bacterial growth <i>in planta</i> of <i>Pst</i> DC3000, ΔCEL/EV, ΔCEL/<i>Pp</i>EC23_ns or ΔCEL/truncated constructs. Diagrams of <i>Pp</i>EC23_ns or truncated constructs of <i>Pp</i>EC23 are provided to the right of the graph. Initial inoculum was adjusted uniformly to 10⁵ CFU/mL. Numbers of bacteria were evaluated at 0 dpi and 4 dpi. C. Transcript level fold change of immune marker genes <i>PR1a</i>, <i>PR2</i>, <i>WRKY12</i> and <i>PI1</i> in leaves of <i>N</i>. <i>benthamiana</i> stably transformed with EV (white bars) or FLAG-<i>Pp</i>EC23_ns (gray bars) at 6 hpi with <i>P</i>. <i>fluorescens</i> strain EtHAn. <i>NbAct1</i> was used as the internal reference gene. <i>T</i>-tests were performed for each comparison. The corresponding <i>P</i> value is shown in the figure. Four biological and four technical replicates were performed.</p

<i>Pp</i>EC23 interacts with itself.

<p>A. Y2H assay showing that <i>Pp</i>EC23 interacts through the C-terminal CM. SD/X/-LWH, SD/-LWHA and SD/-LW represent SD/X-α-gal (-Leu/-Trp/-His), SD (-Leu/-Trp/-His/-Ade) and SD (-Leu/-Trp), respectively. The structural diagrams of <i>Pp</i>EC23_ns or truncated constructs of <i>Pp</i>EC23 are provided next to the relevant strain. The interaction of murine p53 (<i>p53</i>) and SV40 large T-antigen (<i>T</i>) was used as a positive control for the system, and human lamin C (<i>lam</i>) was used as a negative control. B. BiFC assay showing that <i>Pp</i>EC23 interacts with itself <i>in planta</i>. YFP, yellow fluorescent protein epifluorescence. BF, bright field. DAPI signal was used as a nuclear marker. Arrows indicate nuclei. Representative images are shown (n ≥ 20). Bar = 20 μm.</p