• apoplastic proteins
• effectors
• fungal pathogen
• plant immunity
• Tet-On
• Ustilago maydis

Being the first cause of economic losses in crops and having evolved different lifestyles and diverse mechanisms to interact with and colonize their hosts, plant fungal pathogens are constituting not only an economically, but also an ecologically significant threat for plants, especially for the cultivated ones. Long term, to the final goal of assuring crop protection, it is important to study plant fungal pathogens biology to understand and counteract fungal strategies for host plant infection and colonization. The fungus Ustilago maydis that parasitizes maize (Zea mays) and its progenitor teosinte (Euchlaena mexicana), causing symptoms on all aerial parts of the plant, resulting in the corn smut disease, it’s considered a model for studying plant-pathogenic fungi interactions, in particular the biotrophic ones. In fact, U. maydis facultative biotrophic nature allows in vitro propagation, enabling molecular manipulation, efficient reverse genetic approaches and genetic engineering. In addition, plant colonization is rapidly reproducible by infecting maize seedlings, completing the infection cycle in circa 2 weeks and the genome is compact (20.5 Mb), owing only 6.7% of repetitive elements, showing little redundancy.
The success of a pathogen invasion is determined at the very early stages of infection, in fact a prerequisite for pathogenicity is the immediate suppression of the host immune system, to allow a consequent fast growth rate upon penetration.
After penetration of the plant cell wall, the Ustilago maydis causes the plant plasma membrane to invaginate, enlarging the surface area between the plant cell and itself, generating a space in the apoplast, called biotrophic interface. At this interface, pathogen and host secrete an array of small molecules and proteins to interfere with their adversary molecular machinery, to counteract respectively plant defense responses and the establishment of pathogen colonization.
The fungal molecules employed to hamper or suppress plant defense responses and to rearrange plant signaling and metabolism are called effectors and they are mainly small proteins ( 300 amino acids), highly induced upon the contact with the host plant.
After secretion into the apoplast, the so-called apoplastic effectors fulfil their function in this space, at the plant-pathogen interface, by guarding the fungal cells by shielding microbial surface, chelating fungal elicitors or by targeting apoplastic plant proteins. Other effectors, classified as symplastic, are further translocated inside the plant cell, where they function in the cytoplasm, in the nucleus or in other cellular compartments. These effectors can interfere with gene expression, inhibit programmed cell death (PCD) or they can alter hormone homeostasis, protein secretion and degradation.
This study aimed to develop a new method to characterize significant plant protein - effector protein interactions, possibly occurring in maize apoplast during Ustilago maydis infection, to exploit a system as close as possible to the native one, avoiding completely heterologous approach such as yeast (Saccharomyces cerevisiae) or Nicotiana tabacum. To this intent, a Tet-On (Tetracycline-controlled gene expression) based inducible system and an overexpression system were established in Ustilago maydis, to let the fungal cells express and secrete plant apoplastic proteins, known to be highly induced during the infection, to screen for their potential antifungal activity. To find which effectors can rescue the fungus from this plant defense, targeting the proteins that showed an antifungal activity, a library of 300 effectors candidates was generated, using a new self-replicating pNEB based vector, to allow the constitutive secretion of the effectors in axenic culture, to screen for survival or no growth inhibition.