Maize stigmas react differently to self- and cross-pollination and fungal invasion

Abstract

During sexual reproduction in flowering plants, tip-growing pollen tubes travel from the stigma inside the maternal tissues of the pistil toward ovules. In maize (Zea mays L.), the stigma is highly elongated, forming thread-like strands known as silks. Only compatible pollen tubes successfully penetrate and grow through the transmitting tract of the silk to reach the ovules. Like pollen, fungal spores germinate at the surface of silks and generate tube-like structures (hyphae) penetrating silk tissue. To elucidate commonalities and differences between silk responses to these distinctive invading cells, we compared growth behavior of the various invaders as well as the silk transcriptome after self-pollination, cross-pollination, and infection using 2 different fungi. We report that self-pollination triggers mainly senescence genes, whereas incompatible pollen from Tripsacum dactyloides leads to downregulation of rehydration, microtubule, and cell wall-related genes, explaining the slower pollen tube growth and arrest. Invasion by the ascomycete Fusarium graminearum triggers numerous defense responses including the activation of monolignol biosynthesis and NAC as well as WRKY transcription factor genes, whereas responses to the basidiomycete Ustilago maydis are generally much weaker. We present evidence that incompatible pollination and fungal infection trigger transcriptional reprograming of maize silks cell wall. Pathogen invasion also activates the phytoalexin biosynthesis pathway.

Figure 1.

Invasion of papilla hair cells and silks of maize by own and alien pollen tubes as well as by 2 different fungi. A) Experimental setup: maize silks were pollinated with Z. mays B73 (self-pollination) and T. dactyloides (incompatible pollination), respectively, and samples collected at 8HAP. Additionally, silks of maize plants were infected with the facultative hemibiotrophic pathogen F. graminearum and the biotrophic pathogen U. maydis. Samples were collected at 7DAI. B) to E) A GFP-labeled maize pollen tube germinated at the apex of a papilla hair cell structure and grows through the papilla (B) in the intercellular space between hair cells (C) while pushing hair cells apart (D) and growing straight inside the silk intracellularly toward the transmitting tract (right) after reaching into the bottom of the hair cell structure (E). Arrowheads indicate cell walls. F) to I) GFP-expressing F. graminearum hyphae invade silks either after penetration of papilla hairs and intercellular growth like pollen tubes (F) or after initial growth at the silk surface (G). Growth inside the silk occurs first intercellular by pushing cell walls apart (H) before cells are penetrated and die (I) Arrowheads indicate cell walls. J) to M) GFP-labeled U. maydis growth through the silk “ignoring” cell walls. An appressorium is visible at the surface of a papilla hair (J) and further fungal growth occurs intracellularly (K and L) Cell wall material is always detectable surrounding intracellular growing fungal hyphae (M) The tip of hyphae is marked by an asterisk. The white color in all images results from propidium iodide counterstaining. Bars: 10 μm.

Figure 2.

Transcriptome relationships between pollinated and infected maize silks. A) PCA of RNA-seq data from the 11 tissue samples used in this study (see Supplementary Table S1 for sample overview). Three biological replicates were used for each sample that include various pollinated, infected, and control samples as indicated. B) Radar (spider) chart displaying the percentage of transcripts from the maize genome expressed in each tissue. C) Heat map of the whole maize genome in response to self-pollination, incompatible pollination, and fungal infection. D) Genes involved in sucrose synthase and E) nucleotide sugar interconversion are strongly regulated after invasion of foreign pollen tubes and fungal hyphae, respectively.

Figure 3.

Comparison of transcriptional response in maize silks after compatible and incompatible pollination. A) Comparison of DEGs between self-pollinated (compatible) and T. dactyloides pollinated (incompatible) silks. The pollen tube transcriptome was included as a control to identify silk-responsive genes to pollination. B) Scatter plot representing log₂ (fold change expression) for common DEGs in self-pollinated maize (x axis) vs. maize pollinated with T. dactyloides (y axis). Network analysis of downregulated DEGs after C) compatible and D) incompatible pollination of maize silks, respectively.

Figure 4.

Self-pollination alters expression of senescence-associated transcription factor genes in maize silks. A) Self and incompatible pollination induce large transcriptional expression as showed in the scatter plot representing the log₂ (fold change expression) for all DEGs in self-pollinated maize (x axis) vs. maize silks pollinated with T. dactyloides (y axis). B) Fold change expression changes of senescence-associated transcription factor genes.

Figure 5.

In contrast to self-pollination, incompatible pollination and fungal invasion alter cell wall–related genes families in maize silks. Fold change expression analysis of gene families related to cell wall formation is shown. Genes encoding cellulose synthases (A) and most genes for cellulose-like synthases (B) are downregulated. Genes encoding glycosyltransferases (C) are differentially expressed, while genes involved in arabinogalactans (D) are downregulated and polygalacturonases (E) are mostly upregulated. Genes for glycosyl hydrolases (F) show a differential expression pattern.

Figure 6.

Genes involved in monolignol biosynthesis are highly upregulated after F. graminearum infection in maize silks. A) Overview of the H-/G-/S-monolignol biosynthesis pathway including products, educts, and respective enzymes. PALs, cinnamate 4-hydroxylases (C4H), 4-coumarate CoA ligases (4CL), hydroxycinnamoyl-CoA transferases (HCT), p-coumarate 3-hydroxylases (C3H), Caffeoyl-CoA 3-O-methyltransferase (CCoAOMT), cinnamoyl-CoA reductases (CCR), ferulate 5-hydroxylase (F5H), caffeic acid O-methyltransferases (COMT), CADs, LAC, peroxidase (PRX), syringyl (S), guaiacyl (G), and p-hydroxyphenyl (H) lignin. Expression pattern of monolignol biosynthesis genes in maize silks including genes encoding B) PAL, C) C4H, D) 4CL, E) C3H, F) HCT, G) COMT, H) CCR, and I) CAD.

Figure 7.

Genes encoding enzymes that facilitate reactive oxygen species reduction and lignin polymerization are only regulated in maize silks by foreign invaders. A) Peroxidase (PRX) and B) LAC genes are largely upregulated after F. graminearum infection, partly upregulated after U. maydis infection and tendentially downregulated by pollen tubes of T. dactyloides.

Figure 8.

NAC and especially WRKY transcription factor genes are induced after fungal infection in maize silks. A) Heat map displaying induction of NAC transcription factor genes in maize silks at indicated conditions. B) Transcript per million reads (TPMs) of selected NAC transcription factor genes. C) Heat map displaying induction of WYRK transcription factor genes in maize silks at indicated conditions. D) TPMs of selected WYRK transcription factor genes indicate high induction especially after F. graminearum infection.

Figure 9.

Activation of the defense system in maize silks to inhibit fungal growth. A) Scatter plot representing the log₂ for all DEGs in the maize silks infected with F. graminearum (x axis) vs. infected with U. maydis (y axis) and B) comparison of corresponding DEGs. C) Overview of the biosynthetic pathway of diterpenoids (zealexins, kauralexins, and dolabralexins), major components of the maize defense system, and the growth hormone GA. Class I diTPSs are shown in pink, Class II diTPSs in green, and P450 enzymes in orange and cyan, respectively. Heat map represent pattern of expression being red high expression and blue low expression. Geranylgeranyl diphosphate (GGPP), ent-CPP, F. graminearum (F), and U. maydis (U). Fold change expression analysis of the various diterpenoid biosynthesis enzyme genes, namely D)ent-CPP synthase (AN2), E)ent-kaurene synthase2 (KSL2), F)ent-kaurene synthase4 (KSL4), G) cytochrome P450 monooxygenase (CYP71Z18), H) diTPSs (TPS6/11), I)ent-kaurene synthase5 (KSL5), and J)ent-kaurene oxidase (KO1).

Figure 10.

Summary of invasion pattern and major transcriptional responses of maize silks to own and alien pollen tubes as well as to 2 different types of fungi. Ocher, own maize pollen tubes; brown, alien T. dactyloides pollen tubes; green, F. graminearum; and blue, U. maydis hyphae. Enlargements show germinated pollen (left) and fungal spores/conidia (right). PH, papilla hair cell; EP, epidermis; GT, ground tissue; VT, vascular tissue; TT, transmitting track. Green arrows represent upregulation. Red arrows depict downregulation. Solid arrows indicate strong response. Unfilled arrows show mild response.