Spatial regulation of immunity: unmasking the secrets of abaxial immunity to powdery mildew

Abstract

This article comments on:

Wu Y, Sexton WK, Zhang Q, Bloodgood D, Wu Y, Hooks C, Coker F, Vasquez A, Wei C-I, Xiao S. 2024. Leaf abaxial immunity to powdery mildew in Arabidopsis is conferred by multiple defense mechanisms. Journal of Experimental Botany 75, 1465–1478.

In the interaction between plants and powdery mildew (PM) fungi, where epidermal cells serve as a battleground, the lower leaf surface (the abaxial side) shows a mysteriously higher immunity than does the upper (adaxial) side. While the abaxial and adaxial leaf surfaces exhibit clear physical distinctions and micro-environmental variations, the extent to which defense signaling pathways contribute to spatial immunity on the abaxial surface remains largely unknown. In this issue, Wu et al. (2024) investigated the genetic and molecular mechanisms underlying leaf abaxial immunity in Arabidopsis. Through a comprehensive analysis of diverse Arabidopsis immune mutants, the authors unveiled that abaxial immunity in Arabidopsis arises from more active glucosinolate metabolism and EDS1/PAD4-dependent defense within the abaxial epidermal layer. This study offers valuable insights into the spatial regulation of plant immunity, and sheds light on strategies to combat pathogens in crops.

Plants face challenges from various pathogenic microorganisms throughout their growth season. Among them, powdery mildew (PM), caused by Ascomycetes in the order of Erysiphales, is a widespread fungal disease characterized by the appearance of white powdery symptoms on above-ground plant organs (Kuhn et al., 2016). In response to attack from various pathogens, plants have developed a range of physical and molecular characteristics that together constitute the inherent immune system of plants. At the phenotypic level, plants exhibit diverse degrees of spatially and temporally distinct immunity to pathogens, a phenomenon observed across various maturation stages and in different organs. An intriguing phenomenon referred to as abaxial immunity highlights a more robust defense against powdery mildew on the lower (abaxial) side of a leaf compared with the upper (adaxial) side (Wu et al., 2024).

In this issue, Wu et al. (2024) reported the genetic and molecular mechanisms underpinning the leaf abaxial immunity in Arabidopsis. Using an easy taping method to fix the position of leaves, the authors observed that the PM infection in the abaxial side was much weaker than that in the adaxial side in Col-0 and two accessions susceptible to PM strain Golovinomyces cichoracearum (Gc) UCSC1. By examining Arabidopsis mutants involved in various immune pathways, the authors discovered that the abaxial immunity in Arabidopsis stems from elevated basal resistance governed by the EDS1/PAD4- and PEN2/PEN3-dependent defenses in the abaxial epidermis of a leaf. This heightened resistance is likely to be in part attributed to high levels of EDS1 and PEN2 proteins within the abaxial epidermal layer.

Strengthened basal defense contributes to abaxial immunity

Abaxial immunity has long been theorized to arise from specialized physical defenses, such as variations in trichome density and the chemical makeup of epicuticular waxes, or unique microenvironmental factors such as humidity (Fig. 1A). By flipping and fixing leaf position with tape, Wu et al. (2024) minimized the impact of differences in micro-environmental conditions. Then, by employing Arabidopsis mutants with impaired trichome development, they observed that a reduction in trichome density did not make a noteworthy contribution to increased susceptibility to powdery mildew. After carefully monitoring different stages of fungal development, Wu et al. found that, although spore germination was comparable on both leaf surfaces, hyphal growth within the first 24 h was significantly lower in the abaxial layer. Notably, the abaxial layer exhibited fewer functional haustoria, leading to poor fungal growth and rare sporulation. The observed abaxial immunity in Col-0 appears to be primarily associated with post-penetration resistance, occurring at an early stage, possibly before or during haustorium biogenesis (Wu et al., 2024).

Fig. 1.

Leaf polarity and its impact on abaxial immunity to powdery mildew (PM). (A) Schematic diagram of morphological traits associated with the abaxial and adaxial side of a leaf. Note the different patterns of PM penetration on the adaxial and abaxial side. Created with BioRender.com. (B) The adaxial and abaxial sides of an Arabidopsis leaf and the regulatory network underlying polarity development. SAM, shoot apical meristem; see text for further details. (C) Spatial strengthening of PEN2/PEN3- and EDS1/PAD4-mediated defense in the abaxial side. The intensity of blue shading indicates the protein level of PEN2 or EDS1. See text for details.

To further investigate the genetic mechanisms underlying leaf abaxial immunity, Wu et al. compared PM spore production when inoculated on the adaxial or abaxial sides in various immune mutants. They used mutants defective in salicylic acid (SA) signaling and/or biosynthesis (eds1, pad4, and sid2), cell wall-based resistance pathways (pen1), glucosinolate metabolism (pen2 and pen3), jasmonic acid (JA) biosynthesis (dde2-2), and ethylene signaling (ein2-1) (Alonso et al., 1999; von Malek et al., 2002; Collins et al., 2003; Lipka et al., 2005; Stein et al., 2006; Zhang et al., 2018). They found that mutants such as dde2 and ein2 did not affect abaxial immunity, suggesting that JA and ethylene play either no role or a minimal role in bolstering immunity on the abaxial side. In contrast, mutants in the PEN2/PEN3 and EDS1/PAD4 pathways exhibited weakened abaxial immunity, indicating the bolstering roles of PEN2/PEN3 and EDS1/PAD4 pathways in abaxial immunity. The genetic data correlated well with the direct quantification of glucosinolates in Arabidopsis leaves, as determined using the matrix-assisted laser desorption-ionization (MALDI) technique (Shroff et al., 2015). The abaxial surface of Arabidopsis leaf contained approximately 50 pmol mm−2 of glucosinolates, whereas its concentration on the adaxial side was 15–30 times lower. In addition, higher concentrations of glucosinolates were found in the midrib and edges of the leaf, aligning with the midrib resistance observed by Wu et al. (2024). Consistently, the accumulation of PEN2 and EDS1 proteins was higher in abaxial epidermal cells than in adaxial epidermal cells. Moreover, using higher order mutants combining EDS1/PAD4/SID2 with PEN1/PEN2/PEN3 mutations, Wu et al. (2024) demonstrated that EDS1/PAD4- and PEN2/PEN3-based defense plays additive roles in abaxial immunity. However, the infection level on the abaxial side was still significantly lower than that on the adaxial side in the same combinatorial genetic background. These genetic data thus suggest that increased activity of these two defense pathways largely, but not fully, contribute to leaf abaxial immunity against powdery mildew in Arabidopsis, with unidentified components accounting for the remaining level of abaxial immunity.

Notably, strong leaf abaxial immunity as in Arabidopsis was observed only in hemp (Cannabis sativa). In contrast, tomato, tobacco, sow thistle, strawberry, squash, and barley showed variable abaxial immunity, sometimes with visible sporulation on their abaxial surfaces by adapted PM fungi, emphasizing that different plant species may have unique spatial configurations for expressing defensive genes in both sides of their leaves. The EDS1/PAD4 pathway is deeply conserved, but key components are lost in several plant lineages (Baggs et al., 2020). The variability in abaxial immunity observed in this study may be attributable to the gain or loss of components in the EDS1/PAD4 signaling pathway, along with potential variations in their regulation in different plant species.

Polarity development and potential crosstalk with immunity

The processes and components of the regulatory network governing leaf polarity development are well understood (Byrne, 2006; Du et al., 2018). After their initiation from the shoot apical meristem (SAM), leaf primordia in most plants inherently undergo polarity development along three spatial axes: the adaxial–abaxial, proximal–distal, and medial–lateral axes (Fig. 1B). The adaxial domain enriches the expression of genes such as ASYMMETRIC LEAVES2 (AS2), members of the HD-ZIP III family, and transcripts of trans-acting siRNAs targeting AUXIN RESPONSE FACTOR (tasiR-ARF), which specify adaxial traits such as trichomes, epidermal cell shape, palisade mesophyll, and xylem differentiation. In contrast, the development of abaxial features is governed by the accumulation of AUXIN RESPONSE FACTOR 3 and 4 (ARF3 and ARF4), KANADIs (KAN), and miR165/166 transcripts (Fig. 1B) (Du et al., 2018). The balanced development of a leaf blade is determined by the antagonistic interaction between abaxial and adaxial genes (Fig. 1B).

Wu et al. (2024) observed higher signal intensity of EDS1–yellow fluorescent protein (YFP) and PEN2–green fluorescent protein (GFP) in the abaxial epidermal cells than in the adaxial cells (Fig. 1C). This observation raised an interesting question regarding the potential link between polarity development and EDS1/PEN2 expression. Although these two processes may appear distinct, there exists an overlap in the genetic regulatory networks that govern them. ASYMMETRIC LEAVES1 (AS1) showcases a versatile role, intricately weaving between leaf polarity development and plant immunity. AS1, together with its partner AS2, specifies the adaxial domain traits by suppressing the expression of KAN and ARF3/4. Interestingly, AS1 also acts as a negative regulator in inducible resistance and a positive regulator in extracellular defenses against bacterial pathogens. Its distinct involvement in phytopathogen responses, independent of AS2, highlights the intricate intersection between leaf development and immunity pathways (Nurmberg et al., 2007). HYPONASTIC LEAVES1 (HYL1), a nuclear dsRNA-binding protein involved in miRNA biogenesis, directs leaf flattening through the modulation of miR165/166, miR319a, and miR160 (Liu et al., 2011). On the other hand, HYL1 emerges as a key contributor to both pathogen-associated molecular pattern (PAMP)- and effector-triggered immune responses in Arabidopsis, showcasing its multifaceted involvement in plant polarity development and immunity (Kwon, 2016). MicroRNA156 (miR156) regulates leaf morphology through targeting the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors (Wu et al., 2009), which specify polarity traits such as abaxial trichome in juvenile leaves. Recent work revealed that SPL10 mediates an age-dependent augmentation of the SA pathway partially by directly promoting PAD4 expression (Hu et al., 2023). REVOLUTA (REV), a member of the HD-ZIP III family, oversees adaxial growth in the early stage of leaf development. Moreover, it serves as a pivotal regulatory node in plant development and immunity by interacting with WRKY53, contributing to the modulation of SA metabolic pathways (Bresson et al., 2022). Taken together, the molecular mechanism for abaxial immunity may be embedded in the regulatory network for leaf morphogenesis.

Future perspectives in unraveling spatial plant–pathogen interactions

The research by Wu et al. (2024) elucidated the enigmatic pre-programmed higher immunity levels in abaxial epidermal cells, characterized by elevated basal expression of both PEN2/PEN3- and EDS1/PAD4-dependent modules. This renders abaxial cells more resistant to PM compared with adaxial peripheral epidermal cells, highlighting the complexity of spatial regulation of innate immunity in plants. Defects in establishing proper polarity in leaf development lead to severe abnormalities, turning a flat leaf into a tiny symmetric structure often with ecotopic expression of meristemic genes. Such defects greatly hinder the assessment of disease phenotypes. Recent breakthroughs employing single-cell RNA sequencing (scRNA-seq) technology offer unprecedented insights into plant–microbe interactions at a single-cell resolution. Exploration of the transformative potential of scRNA-seq has helped decipher intricate transcriptional dynamics and spatial heterogeneity in cell type-specific responses (Tang et al., 2023; Zhu et al., 2023). The expression of EDS1–YFP and PEN2–GFP is higher in the abaxial epidermal cells than in the adaxial cells (Fig. 1C). It is interesting to explore whether such a spatial difference was regulated at the transcriptional or post-transcriptional level. Furthermore, it would be intriguing to assess the accumulation of EDS1 and PEN2 in polarity mutants. Thus, future research fine-tuning the level of EDS1 and PEN2 by fusing them to adaxial or abaxial promoters may further elucidate the significance of this unbalanced expression pattern. The pre-programmed higher immunity in the abaxial surface suggests that mild elevation of the PEN2/PEN3- and EDS1/PAD4-dependent defense may have limited negative impact on plant growth, shedding light on potential engineering strategies with balanced growth–defense trade-off.

Conflict of interest

The authors declare no conflict of interest.

Funding

LY is supported by National Institutes of Health (NIH) R35GM143067.