Soybean homologs of MPK4 negatively regulate defense responses and positively regulate growth and development

Abstract

Mitogen-activated protein kinase (MAPK) cascades play important roles in disease resistance in model plant species such as Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum). However, the importance of MAPK signaling pathways in the disease resistance of crops is still largely uninvestigated. To better understand the role of MAPK signaling pathways in disease resistance in soybean (Glycine max), 13, nine, and 10 genes encoding distinct MAPKs, MAPKKs, and MAPKKKs, respectively, were silenced using virus-induced gene silencing mediated by Bean pod mottle virus. Among the plants silenced for various MAPKs, MAPKKs, and MAPKKKs, those in which GmMAPK4 homologs (GmMPK4s) were silenced displayed strong phenotypes including stunted stature and spontaneous cell death on the leaves and stems, the characteristic hallmarks of activated defense responses. Microarray analysis showed that genes involved in defense responses, such as those in salicylic acid (SA) signaling pathways, were significantly up-regulated in GmMPK4-silenced plants, whereas genes involved in growth and development, such as those in auxin signaling pathways and in cell cycle and proliferation, were significantly down-regulated. As expected, SA and hydrogen peroxide accumulation was significantly increased in GmMPK4-silenced plants. Accordingly, GmMPK4-silenced plants were more resistant to downy mildew and Soybean mosaic virus compared with vector control plants. Using bimolecular fluorescence complementation analysis and in vitro kinase assays, we determined that GmMKK1 and GmMKK2 might function upstream of GmMPK4. Taken together, our results indicate that GmMPK4s negatively regulate SA accumulation and defense response but positively regulate plant growth and development, and their functions are conserved across plant species.

Figure 1.

Silencing GmMPK4 constitutively activates defense responses in soybean plants. A, Stunted stature. B, Purple necrosis on the stem. C, Symptoms of the empty BPMV vector (BPMV-0) on a trifoliolate leaf. D, Rough leaves and purple pigmentation in the veins compared with vector control plants. E, Spontaneous cell death on the leaves of GmMPK4-silenced plants at 20 dpi. F, RT-PCR showing that the transcript levels of GmMPK4 and GmPR2 were reduced and induced, respectively, on the leaves of GmMPK4-silenced plants. GmElF1b served as a control. The results shown are from four individual GmMPK4-silenced plants (BPMV-GmMPK4) and BPMV vector control plants (BPMV-0). G, RT-PCR showing that GmMPK4c/4d was silenced, whereas GmMPK3 and GmMPK6 were not silenced, in GmMPK4-silenced plants.

Figure 2.

Overaccumulation of both SA and H₂O₂ in GmMPK4-silenced soybean plants. A, Both free SA and bound SA (SAG) levels were measured in GmMPK4-silenced and vector control plants at 20 dpi. Error bars represent sd for five independent samples; ** P < 0.01, Student’s t test. FW, Fresh weight. B, The presence of H₂O₂ in the soybean leaves was visualized by staining with DAB. Oxidized DAB forms a reddish-brown deposit.

Figure 3.

Examples of up- and down-regulated genes or gene families in GmMPK4-silenced plants versus vector control plants. Fold changes (log₂) of gene expression values in GmMPK4-silenced plants versus vector control plants (q < 0.01) are shown. Blue indicates higher expression in GmMPK4-silenced plants, whereas red indicates higher expression in vector control plants. The diagrams were generated using the pathway analysis program MapMan (https://gabi.rzpd.de/projects/MapMan/; Thimm et al., 2004). Each square represents the fold change of one gene. Numbers +4.5 to –4.5 on the color scale represent relative log₂ fold change. The minimum log₂ fold change is 1 or –1.

Figure 4.

Silencing GmMPK4s enhances the resistance of soybean plants against SMV. At 18 dpi with BPMV constructs, SMV-N-GUS was biolistically delivered into detached leaves of either BPMV-0 or BPMV-GmMPK4 plants. The number and diameter of GUS foci were determined at 3 dpi with SMV-N-GUS. A, Comparison of infection foci of SMV-N-GUS on the leaves of BPMV-0 and BPMV-GmMPK4 plants. B, Comparison of the number of SMV-N-GUS foci on the leaves of BPMV-0 and BPMV-GmMPK4 plants. C, Closeup images of GUS foci shown in A with a dissecting microscope. Bars = 2 mm. D, Comparison of the size of SMV-N-GUS foci on the leaves of BPMV-0 and BPMV-GmMPK4 plants. The data shown in B and D are mean values of four individual leaves from four different plants. Error bars in B and D represent sd for four independent leaves. At least 30 GUS foci from each of four independent leaves were measured in D. ** P < 0.01, Student’s t test.

Figure 5.

Silencing GmMPK4s enhances the resistance of soybean plants against downy mildew. A and B, Chlorotic lesions typical of soybean downy mildew were detected on the leaves of vector control plants (A) but not on the leaves of GmMPK4-silenced plants 1 week after inoculation with P. manschurica (B). C and D, P. manschurica hyphae were observed in the mesophyll of vector control plants (C) but not in the mesophyll of GmMPK4-silenced plants 1 week after inoculation with P. manschurica (D). E, Germ tubes (GT) with multiple appressoria that were not able to penetrate the epidermal surface were often observed on GmMPK4-silenced plants. 1, Sporangium; 2 to 4, appressoria. Bars = 150 μm in C and D and 80 μm in E.

Figure 6.

GmMPK4a is localized to both cytoplasm and nucleus. The GFP-GmMPK4a and free DsRed constructs driven by the 35S promoter were cobombarded into onion epidermal cells. The left panel shows the transient expression of GFP-GmMPK4a in onion cells; the middle panel shows the transient expression of free RFP in onion cells; and the right panel shows the merged image. Bar = 100 μm.

Figure 7.

Both GmMKK1 and GmMKK2 interact with GmMPK4a. YFP epifluorescence (left panels), bright-field (middle panels), and merged (right panels) images of onion epidermal cells cobombarded with constructs expressing different fusion proteins as indicated are shown. Coexpression of nYFP-AtbZIP and cYFP-AtbZIP was used as a positive control, and cobombardment of nYFP-GmMKK4 and cYFP-GmMPK4a was used as negative control. Bar = 100 μm.

Figure 8.

GmMKK1 and GmMKK2 phosphorylate GmMPK4a in vitro. The top panel is an autoradiograph of the phosphorylation assay, and the bottom panel is the same SDS gel stained with Coomassie Brilliant Blue. Lanes are as follows: MBP-GmMPK4a alone (lane 1), MBP-GmMKK1 alone (lane 2), MBP-GmMKK2 alone (lane 3), MBP-GmMPK4a + MBP-GmMKK1 (lane 4), and MBP-GmMPK4a + MBP-GmMKK2 (lane 5).