Project 2A: Mechanochemical Signaling in Plant Immune Responses
Project 2A determines how cells communicate and integrate mechanical and chemical signals by studying how plant cells sense microbial pathogens and organize a defense response. Objectives include: (1) quantitatively analyze Ca2+ signaling and determine how it functions upstream of actin dynamics or cytoskeletal remodeling during the plant defense response elicited by a local or global PAMP/DAMP stimulus; (2) identify plant mechanoreceptors involved in perception of fungal/oomycete attack and determine their role in Ca2+ signaling of actin organization/dynamics; and (3) investigate the impact of bacterial effector proteins on cytoskeletal organization in tobacco leaves, specifically focusing on RipU's effects on actin filaments and cortical microtubules.
Research Outputs
Local traveling waves of cytosolic calcium elicited by defense signals or wounding are propagated by distinct mechanisms
Live-cell imaging and modeling revealed that MAMP-induced calcium waves in plants propagate via distinct mechanisms from mechanical damage.
Fig. 1. MAMP-induced Ca²⁺ signatures and cell-to-cell signal propagation follow a defined spatial pattern.
(A) A representative timelapse image series of R-GECO1 fluorescence dynamics in Arabidopsis cotyledon epidermal cells following global treatment with 1 µM flg22. See also Supplemental Movie S1. Cytosolic Ca²⁺ elevations were first detected in initiator cells and then propagated to neighboring cells. Bar = 50 µm. (B) Annotation of initiator cells (N), N+1 and N+2 neighboring cells shown in (A). (C) Representative traces for the Ca²⁺ intensity ratio of an N, N+1, and N+2 cell, respectively. Additional representative traces are shown in Figure S5. Fluorescence intensity changes were calculated as the ratio ΔF/F₀. Black dashed lines indicate the time when flg22 was added. (D) Quantitative analysis of peak features for Ca²⁺ traces from N, N+1 and N+2 cells. Each data point in the box plots represents an average value measured from 5–15 cells from a single cotyledon; data from 8 cotyledons from 3 independent experiments denoted by different shapes are presented in each box plot. One-way ANOVA and Tukey’s HSD test, different letters indicate significant differences with P < 0.05.
Zhang, W., N. Kumar, J. Helwig, A. Hoerter, A.S. Iyer-Pascuzzi, D. Umulis, E. Pienaar, and C.J. Staiger. 2025. Local traveling waves of cytosolic Ca2+ elicited by defense signals or wounding are propagated by distinct mechanisms in Arabidopsis. Science Signaling, 18(915), eadw2270.
Cooperative actin filament nucleation by the Arp2/3 complex and formins maintains the homeostatic cortical array in Arabidopsis epidermal cells
Discovered that Arp2/3 and formins cooperatively maintain actin array architecture in plant cells, with unexpected compensatory nucleation upon dual inhibition.
Figure 1.
Genetic disruption of the Arp2/3 complex leads to reduced actin filament density and bundling. A) Representative images of epidermal cells from the apical region of 5-d-old etiolated hypocotyls expressing GFP-fABD2 imaged by VAEM are shown in the left columns. Scale bar: 20 μm. ROIs (boxes) were magnified and shown in the right columns. Scale bar: 5 μm. B to D) Quantitative analysis of the percentage of occupancy or density of actin filament arrays B) and the extent of filament bundling as measured by skewness C) and coefficient of variance D) analyses. Both the density and the bundling of actin arrays in arp2-1 and arpc2 cells were significantly decreased compared to those in the respective wild-type cells. In box-and-whisker plots, boxes show the interquartile range and the median, and whiskers show the maximum–minimum interval of 3 biological repeats with independent populations of plants. Individual biological repeats are represented with different shapes (n = 30 seedlings, 10 seedlings per biological repeat). Letters a and b denote groups that show statistically significant differences with other genotypes by 1-way ANOVA with Tukey’s post hoc test (P < 0.05). WT, wild type.
Xu, L., L. Cao, J. Li, and C.J. Staiger. 2024. Cooperative actin filament nucleation by the Arp2/3 complex and formins maintains the homeostatic cortical array in Arabidopsis epidermal cells. Plant Cell, 36: 764–789.
A Ralstonia solanacearum type III effector alters the actin and microtubule cytoskeleton to promote bacterial virulence in plants
The bacterial effector RipU was found to disrupt both actin and microtubule networks in plants, enhancing pathogen virulence.
Fig 3. RipUK60 physically associates with the cytoskeleton.
(A) RipUK60-GFP co-immunoprecipitates with actin and tubulin. The indicated constructs were transiently expressed in N. benthamiana leaves. All transgenes were under the control of a 35S promoter. Total protein was isolated 48 hpi, immunoprecipitated by GFP-Trap agarose bead slurry, and immunoblotted with the indicated antibodies. (B) RipUK60 associates with tomato actin in a yeast two hybrid assay. Left, SC-Leu-Trp-His + 10 mM 3AT selection plates, right SC-Leu-Trp-His + 100 mM 3AT. Top two rows of each set of panels show that SlActin and RipU interaction promotes yeast growth when cloned as either bait (pDEST32) or prey (pDEST22). Four lower rows are negative controls with either SlActin or RipU and an empty bait or prey vector. Dilutions of indicated constructs were plated on yeast selective media (SC-Leu-Trp-His +3AT). All experiments were repeated at least three independent times. (C) Co-IP/MS analysis. Immunoprecipitation was done by GFP-Trap agarose bead slurry, and eluants were subjected to mass spectrometry. The volcano plots depict the differential enrichment of proteins between 35S:RipUK60-GFP and 35S:GFP. The blue dots are proteins with p-value ≤ 0.05 and log₂FC > 1. Actin and tubulin proteins are indicated in green and red respectively. Two tubulin alpha proteins and an actin protein with greater than 1.5-fold change (log₂FC > 0.585) are indicated. The result represents three independent experiments.
Hiles R., et al. 2024. A Ralstonia solanacearum type III effector alters the actin and microtubule cytoskeleton to promote bacterial virulence in plants. PLoS Pathogens, 20(12):e1012814.