A novel <i>Meloidogyne graminicola</i> effector, MgGPP, is secreted into host cells and undergoes glycosylation in concert with proteolysis to suppress plant defenses and promote parasitism

<div><p>Plant pathogen effectors can recruit the host post-translational machinery to mediate their post-translational modification (PTM) and regulate their activity to facilitate parasitism, but few studies have focused on this phenomenon in the field of plant-parasitic nematodes. In this study, we show that the plant-parasitic nematode <i>Meloidogyne graminicola</i> has evolved a novel effector, MgGPP, that is exclusively expressed within the nematode subventral esophageal gland cells and up-regulated in the early parasitic stage of <i>M</i>. <i>graminicola</i>. The effector MgGPP plays a role in nematode parasitism. Transgenic rice lines expressing MgGPP become significantly more susceptible to <i>M</i>. <i>graminicola</i> infection than wild-type control plants, and conversely, <i>in planta</i>, the silencing of MgGPP through RNAi technology substantially increases the resistance of rice to <i>M</i>. <i>graminicola</i>. Significantly, we show that MgGPP is secreted into host plants and targeted to the ER, where the <i>N</i>-glycosylation and C-terminal proteolysis of MgGPP occur. C-terminal proteolysis promotes MgGPP to leave the ER, after which it is transported to the nucleus. In addition, <i>N</i>-glycosylation of MgGPP is required for suppressing the host response. The research data provide an intriguing example of <i>in planta</i> glycosylation in concert with proteolysis of a pathogen effector, which depict a novel mechanism by which parasitic nematodes could subjugate plant immunity and promote parasitism and may present a promising target for developing new strategies against nematode infections.</p></div

Assays for glycosylation of MgGPP.

<p>(A) Using an anti-MgGPP antibody, western blot analysis of proteins from pre-J2s, par-J3s/J4s and females of <i>Meloidogyne graminicola</i> treated with or without PNGase F all showed the ~25 kDa size band, indicating that MgGPP is not glycosylated in nematodes. (B) Using an anti-GFP antibody, western blot analysis of proteins from the transformed cells of rice and tobacco showed two protein forms of ~43 and ~39 kDa of eGFP:MgGPP^Δsp, the ~39 kDa size of MgGPP^Δsp:eGFP treated with PNGase A, and ~39 kDa size of the point mutation eGFP:MgGPP^Δsp_N110Q, indicating that <i>N</i>-glycosylation of MgGPP occurred in host plants.</p

<i>In planta</i> RNAi of <i>MgGPP</i> attenuates <i>Meloidogyne graminicola</i> parasitism.

<p>(A) qRT-PCR analysis to detect the GUS intron fragment was used to confirm dsRNA expression levels in roots of RNAi lines. Transgenic RNAi rice lines showed a decreased number of females in roots compared with the controls. The data are presented as the means ± standard deviation (SD) from fifteen plants. (B) qRT-PCR assays of the expression levels of <i>MgGPP</i> in <i>M</i>. <i>graminicola</i> collected from RNAi lines, transgenic empty vector (EV) plants and wild type (WT) plants. The expression levels of <i>Mg-CRT</i> and <i>Mg-expansin</i> from <i>M</i>. <i>graminicola</i> were used to determine the specificity of the <i>MgGPP-</i>targeting RNAi. *P < 0.05; **P < 0.01, Student’s t test. RNAi6, 15, 25 and 26, different transgenic RNAi rice lines.</p

Subcellular localization of MgGPP^{Δsp_Δ123–224} in the rice root protoplast cells.

<p>(A) Schematic diagram showing the protein structures of MgGPP mutants. (B) eGFP:MgGPP^{Δsp_Δ123–224} was transformed into rice root protoplasts. Signals that colocalized with mCherry were observed in the cytoplasm and nuclei at ~8 h after cotransformation. (C) Using an anti-GFP antibody, western blot analysis of proteins from transformed cells showed a ~39-kDa band in cells transformed with eGFP:MgGPP^{Δsp_Δ123–224} and two protein forms of ~43 and ~39 kDa in the cells transformed with eGFP:MgGPP^Δsp. (D) As a control, eGFP:MgGPP^Δsp was transformed into rice root protoplasts. Signals were observed in the ER at ~8 h after cotransformation. These indicated MgGPP lacking amino acids 123–224 could not be imported into the ER and glycosylated. Scale bar, 50 μm.</p

MgGPP localization in sectioned rice root galls at 5 dpi.

<p>(A-D) Localization of the secreted MgGPP protein in the giant cell nuclei (red arrows), the cell wall of adjacent giant cells (white arrows) and the lumen of the anterior esophagus of the nematode (yellow arrows). (E-H) Localization of the secreted MgGPP protein in the giant cell nuclei (red arrows). (I-L) Localization of the secreted MgGPP protein in the giant cell nucleus (red arrow) and the lumen of the anterior esophagus of the nematode (yellow arrows). Micrographs A, E and I are observations of the Alexa Fluor 488-conjugated secondary antibody. Micrographs B, F and J are images of 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei. Micrographs C, G and K are images of differential interference contrast. Micrographs D, H and L are superpositions of images of the Alexa Fluor 488-conjugated secondary antibody, DAPI-stained nuclei and differential interference contrast. N, nematode; asterisks, giant cells; M, metacorpus; H, the head of <i>M</i>. <i>graminicola</i>; Scale bars, 20 μm.</p

Subcellular localization of eGFP:MgGPP^{Δsp_Δ123–224}:HDEL and eGFP:MgGPP^Δsp:HDEL in the rice root protoplast cells.

<p>(A) Schematic diagram showing the protein structures of WAK2ss:eGFP:MgGPP^{Δsp_Δ123–224}:HDEL and WAK2ss:eGFP:MgGPP^Δsp:HDEL. (B) WAK2ss:eGFP:MgGPP^{Δsp_Δ123–224}:HDEL was transformed into rice root protoplasts. HDEL is a signal for retention in the ER, and WAK2ss-mCherry-HDEL was used as a marker to indicate the ER. The NH2-terminal signal sequence (WAK2ss) from <i>Arabidopsis thaliana</i> wall-associated kinase 2 was used to direct fusion proteins to secretory compartments. Signals that colocalized with the ER marker WAK2ss-mCherry-HDEL were consistently observed in the ER after cotransformation. (C) WAK2ss:eGFP:MgGPP^Δsp:HDEL was transformed into rice root protoplasts. Signals that colocalized with the ER marker WAK2ss-mCherry-HDEL were observed in the ER at ~8 h after cotransformation, and signals that colocalized with mCherry were observed in the nuclei at ~48 h after cotransformation. (D) Using an anti-GFP antibody, western blot analysis of proteins from transformed cells showed two protein forms of ~43 and ~39 kDa of both eGFP:MgGPP^{Δsp_Δ123–224}:HDEL and eGFP:MgGPP^Δsp:HDEL. These indicated that the glycosylation of MgGPP occurred in the ER, and proteolysis of MgGPP^123-224 led MgGPP^{Δsp_Δ123–224} to leave the ER. Scale bar, 50 μm.</p

Expression patterns of <i>MgGPP</i> in <i>Meloidogyne graminicola</i>.

<p>(A) Schematic representation of <i>M</i>. <i>graminicola</i> pre-J2. (B) Localization of <i>MgGPP</i> in the subventral esophageal glands of <i>M</i>. <i>graminicola</i> pre-J2s by <i>in situ</i> hybridization. Fixed nematodes were hybridized with (left) sense and (right) antisense cDNA probes from <i>MgGPP</i>. Scale bars, 10 μm. (C) The developmental expression pattern of MgGPP by RT-qPCR analysis in five different life stages of <i>M</i>. <i>graminicola</i>. The fold change values were calculated using the 2^-ΔΔCT method and presented as the change in mRNA level at various nematode developmental stages relative to that of the egg stage. The data shown are the means of three repeats plus standard deviation (SD), and three independent experiments were performed with similar results. dpi, days post-infection; pre-J2, pre-parasitic second-stage juvenile; par-J2, par-J3 and par-J4, parasitic second-, third- and fourth-stage juveniles, respectively.</p

Transgenic lines expressing MgGPP in rice exhibit enhanced susceptibility to <i>Meloidogyne graminicola</i>.

<p>(A) Western blot confirmation of the MgGPP product with an anti-MgGPP antibody. Two protein forms of ~12 and ~ 16 kDa were detected because of the glycosylation and proteolysis of MgGPP. As a control, one protein of ~25 kDa was detected in <i>M</i>. <i>graminicola</i>. (B) qRT-PCR analysis was used to confirm the <i>MgGPP</i> mRNA expression level in transgenic-<i>MgGPP</i> lines. Transgenic rice expressing <i>MgGPP</i> showed an increased number of females in roots compared with the controls. The data are presented as the means ± standard deviation (SD) from fifteen plants. *P < 0.05; **P < 0.01, Student’s t test. OE-4, 5, 6, 9 and 39, five transgenic rice lines; Mg, <i>M</i>. <i>graminicola</i>; EV, empty vector; WT, wild type.</p

Suppression of Gpa2/RBP-1-triggered cell death by MgGPP.

<p>(A) Schematic diagram showing the protein structures of MgGPP mutants. (B) Assay of the suppression of Gpa2/RBP-1<b>-</b>triggered cell death in <i>Nicotiana benthamiana</i> by MgGPP. <i>N</i>. <i>benthamiana</i> leaves were infiltrated with buffer or <i>Agrobacterium tumefaciens</i> cells carrying flag:MgGPP^{Δsp_Δ123–224}, flag:MgGPP^Δsp, GrCEP12, flag:MgGPP^Δsp_N110Q and the flag control gene, followed 24 h later with <i>A</i>. <i>tumefaciens</i> cells carrying the Gpa2/RBP-1 genes. The cell death phenotype was scored and photographs were taken 5 days after the last infiltration. (C) The average areas of cell death of in leaves infected with MgGPP and other proteins followed by Gpa2/RBP-1. The columns with asterisks indicates a highly statistically significant reduction of the necrosis index of MgGPP and GrCEP12 compared with that of the negative control flag. Each column represents the mean with the standard deviation (n = 55). **P<0.01, Student’s t test. (D) RT-PCR confirmation of the expression of <i>MgGPP</i>, <i>RBP-1</i> and <i>Gpa2</i>. (E) Western blot analysis was used to confirm the expression of RBP-1, MgGPP and MgGPP mutants with an anti-GFP antibody.</p

DataSheet1_Mining candidate genes for rice cadmium accumulation in the shoot through a genome-wide association study and transcriptomic analysis.xlsx

High cadmium (Cd) accumulation in rice is a serious threat to human health. The genetic mechanism of Cd accumulation in rice is highly complicated. To identify the low Cd accumulation in rice germplasm, investigate the genetic mechanism underlying Cd accumulation, and mine the elite genes of significant importance for rice breeding of low Cd accumulation varieties, we performed a genome-wide association study (GWAS) for rice Cd concentration in the shoot. The rice accessions were 315 diverse indica rice accessions selected from the 1568 rice accessions with 700,000 SNPs. Within the high rate of linkage disequilibrium (LD) decay, eight QTLs related to rice Cd accumulation were identified. Transcriptomic analysis showed there were 799 differentially expressed genes (DEGs) in the root and 857 DEGs in the shoot, which are probably considered to be the cause of the significant difference in Cd accumulation between high and low Cd accumulation varieties. In qCd11-1, we detected a crucial candidate gene, LOC_Os11g11050, which encodes an initiation factor, expressed differently in the root between the high and low Cd accumulation varieties. Furthermore, under Cd treatment, the expression levels of LOC_Os11g11050 significantly decreased in both the high and low Cd accumulation varieties. Sequence comparison and qRT-PCR revealed that there were indel sequences and base substitutions in the promoter region of LOC_Os11g11050 correlated with the LOC_Os11g11050 expression level, as well as the phenotype of Cd concentration differences in shoot between the high and low Cd accumulation accessions. LOC_Os11g11050 might play important roles in Cd accumulation. The results of our study provide valuable resources for low Cd accumulation in indica varieties and the candidate functional gene, as well as molecular mechanisms for Cd accumulation in indica rice. The genetic architecture underlying Cd accumulation in indica can be used for further applying the low Cd gene existing in indica for decreasing Cd accumulation in rice.</p