Cellulose is synthesized by cellulose synthases (CESAs) from the glycosyltransferase GT-2 family. In plants, the CESAs form a six-lobed rosette-shaped CESA complex (CSC). Here we report crystal structures of the catalytic domain of Arabidopsis thaliana CESA3 (AtCESA3CatD) in both apo and uridine diphosphate (UDP)-glucose (UDP-Glc)–bound forms. AtCESA3CatD has an overall GT-A fold core domain sandwiched between a plant-conserved region (P-CR) and a class-specific region (C-SR). By superimposing the structure of AtCESA3CatD onto the bacterial cellulose synthase BcsA, we found that the coordination of the UDP-Glc differs, indicating different substrate coordination during cellulose synthesis in plants and bacteria. Moreover, structural analyses revealed that AtCESA3CatD can form a homodimer mainly via interactions between specific beta strands. We confirmed the importance of specific amino acids on these strands for homodimerization through yeast and in planta assays using point-mutated full-length AtCESA3. Our work provides molecular insights into how the substrate UDP-Glc is coordinated in the CESAs and how the CESAs might dimerize to eventually assemble into CSCs in plants.

Fusarium oxysporum is a soil-borne fungal pathogen of several major food crops. Research on understanding the molecular details of fungal infection and the plant’s defense mechanisms against this pathogen has long focused mainly on the tomato-infecting F. oxysporum strains and their specific host plant. However, in recent years, the Arabidopsis thaliana - Fusarium oxysporum strain 5176 pathosystem has additionally been established to study this plant-pathogen interaction with all the molecular biology, genetic and genomic tools available for the A. thaliana model system. Work on this system has since produced several new insights, especially in regards to the role of phytohormones involved in the plant’s defense response, and the receptor proteins and peptide ligands involved in pathogen-detection. Furthermore, work with the pathogenic strain Fo5176 and the related endophytic strain Fo47 has demonstrated the suitability of this system for comparative studies of the plant’s specific responses to general microbe- or pathogen-associated molecular patterns. In this review, we highlight the advantages of this specific pathosystem, summarize the advances made in studying the molecular details of this plant-fungus interaction, and point out open questions that remain to be answered.

The regulatory sequences controlling the expression of a gene (i.e. the promoter) are essential to properly understand a gene's function. From their use in mutant complementation assays, to studying their responsiveness to different stimuli via transcriptional reporter lines or using them as proxy for the activation of certain pathways, assays using promoter sequences are valuable tools for insight into the genetic architecture underlying plant life. The GreenGate (GG) system is a plant-specific variant of the Golden Gate assembly method, a modular cloning system that allows the hierarchical assembly of individual donor DNA fragments into one expression clone via a single reaction step. The assembly is based on specific recognition sites for the individual donor fragments, derived i.e., from a promoter, coding sequence, resistance gene or a protein tag. Here, we present a collection of 75 GG entry vectors carrying putative regulatory sequences for Arabidopsis thaliana genes involved in many different pathways of the plant immune system, designated Plant Immune system Promoters (PIP). This pGG-PIP entry vector set enables the rapid assembly of expression vectors to be used for transcriptional reporters of plant immune system components, mutant complementation assays when coupled with coding sequences, mis-expression experiments for genes of interest, or the targeted use of CRISPR/Cas9 genome editing. We used pGG-PIP vectors to create fluorescent transcriptional reporters in A. thaliana and demonstrated the potential of these reporters to image the responsiveness of specific plant immunity genes to infection and colonization by the fungal pathogen Fusarium oxysporum. Using the PLANT ELICITOR PEPTIDE (PEP) pathway as an example, we show this pathway is locally activated in response to colonization by the fungus.

Jasmonic acid (JA), ethylene (ET) and salicylic acid (SA) are the three major phytohormones coordinating a plant's defense response to pathogenic attack. While JA and ET are assumed to primarily control the defense against necrotrophic pathogens, SA-induced defense responses target mainly biotrophic microbes, and can include drastic measures such as programmed cell death as part of the plant's hypersensitive response (HR). Fusarium oxysporum is a hemibiotrophic fungal pathogen of several plant species, including many important food crops, and the model plant species Arabidopsis thaliana. Colonization of the plant's root vascular tissue by the fungus eventually results in wilting and plant death. A general role for JA, ET and SA in combating infection and colonization of the plant by F. oxysporum has been demonstrated, but their distinct roles and modes of action have so far not been described. Here, using high resolution microscopy with fluorescent marker lines of A. thaliana roots infected with F. oxysporum we show that SA acts spatially separate from JA/ET, in a distinct set of root cells immediately neighboring the fungal colonization site. JA and ET, however, act together in a different, but also clearly defined set of cells, slightly removed from the colonization site. SA induces HR in the cells directly in contact with already colonized tissue, that presumably can no longer be rescued from infection, stopping the spread of colonization. In comparison, JA and ET act in a group of cells slightly removed from the HR site, initiating a defense response to actively resist the invader. These results show how the three phytohormones act together, but spatially and functionally separate from each other, to fight this hemibiotrophic pathogen. Such a high-resolution analysis to resolve the plant's immune response to pathogenic infection on an individual cell level and in intact tissue has so far been lacking.

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