Identification and characterization of potato protease inhibitors able to inhibit pathogenicity and growth of Botrytis cinerea

Different tissues of potato, tobacco, and bean plants were screened for anti-fungal protease inhibitor (PI) activity, also following fungal pathogen inoculation or mechanical wounding. A potato (Solanum tuberosum var. Desireé) sprout protein extract showed a strong inhibitory activity against chymotrypsin and Botrytis cinerea fungal proteases, but also on spore germination, hyphal elongation, and development of necrotic lesions. An active mixture of different proteins was affinity column purified and sequenced. Two new anti-fungal genes, PKI1 and PPI3B2, coding, respectively, for a Kunitz-type inhibitor and a Proteinase Inhibitor 1 capable of reducing fungal lesion development, were cloned and partially characterized. Direct effect on leaf necrosis formation was found to be dependent on the anti-chymotrypsin activity of both selected inhibitors. The PKI1 transcript was found to accumulate in untreated sprout tissues, although homologues of this gene seemed to accumulate following Bemisia tabaci attack. In the case of PPI3B2, we provide preliminary evidence that a member of the Proteinase Inhibitor 1 family is active against not only herbivorous insects but also phytopathogenic fungi and foliar lesions caused by them

Cytosolic pH Controls Fungal MAPK Signaling and Pathogenicity

Mitogen-activated protein kinases (MAPKs) regulate a variety of cellular processes in eukaryotes. In fungal pathogens, conserved MAPK pathways control key virulence functions such as infection-related development, invasive hyphal growth, or cell wall remodeling. Recent findings suggest that ambient pH acts as a key regulator of MAPK-mediated pathogenicity, but the underlying molecular events are unknown. Here, we found that in the fungal pathogen Fusarium oxysporum, pH controls another infection-related process, hyphal chemotropism. Using the ratiometric pH sensor pHluorin we show that fluctuations in cytosolic pH (pHc) induce rapid reprogramming of the three conserved MAPKs in F. oxysporum, and that this response is conserved in the fungal model organism Saccharomyces cerevisiae. Screening of a subset of S. cerevisiae mutants identified the sphingolipid-regulated AGC kinase Ypk1/2 as a key upstream component of pHc-modulated MAPK responses. We further show that acidification of the cytosol in F. oxysporum leads to an increase of the long-chain base (LCB) sphingolipid dihydrosphingosine (dhSph) and that exogenous addition of dhSph activates Mpk1 phosphorylation and chemotropic growth. Our results reveal a pivotal role of pHc in the regulation of MAPK signaling and suggest new ways to target fungal growth and pathogenicity.Ministerio de Ciencia e Innovación (España)Junta de AndalucíaDepto. de Microbiología y ParasitologíaFac. de FarmaciaTRUEpu