The disordered C-terminal tail of fungal LPMOs from phytopathogens mediates protein dimerization and impacts plant penetration.

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that oxidatively degrade various polysaccharides, such as cellulose. Despite extensive research on this class of enzymes, the role played by their C-terminal regions predicted to be intrinsically disordered (dCTR) has been overlooked. Here, we investigated the function of the dCTR of an LPMO, called CoAA9A, up-regulated during plant infection by Colletotrichum orbiculare, the causative agent of anthracnose. After recombinant production of the full-length protein, we found that the dCTR mediates CoAA9A dimerization in vitro, via a disulfide bridge, a hitherto-never-reported property that positively affects both binding and activity on cellulose. Using SAXS experiments, we show that the homodimer is in an extended conformation. In vivo, we demonstrate that gene deletion impairs formation of the infection-specialized cell called appressorium and delays penetration of the plant. Using immunochemistry, we show that the protein is a dimer not only in vitro but also in vivo when secreted by the appressorium. As these peculiar LPMOs are also found in other plant pathogens, our findings open up broad avenues for crop protection.

Tandem metalloenzymes gate plant cell entry by pathogenic fungi.

Global food security is endangered by fungal phytopathogens causing devastating crop production losses. Many of these pathogens use specialized appressoria cells to puncture plant cuticles. Here, we unveil a pair of alcohol oxidase-peroxidase enzymes to be essential for pathogenicity. Using Colletotrichum orbiculare, we show that the enzyme pair is cosecreted by the fungus early during plant penetration and that single and double mutants have impaired penetration ability. Molecular modeling, biochemical, and biophysical approaches revealed a fine-tuned interplay between these metalloenzymes, which oxidize plant cuticular long-chain alcohols into aldehydes. We show that the enzyme pair is involved in transcriptional regulation of genes necessary for host penetration. The identification of these infection-specific metalloenzymes opens new avenues on the role of wax-derived compounds and the design of oxidase-specific inhibitors for crop protection.

Lipid droplets in the pheromone glands of bombycids: Effects of larval diet on their size and pheromone titer.

In addition to the blend ratio, the quantity of sex pheromone components secreted by female moths may affect the efficient attraction of conspecific males. The present study using the silkmoth Bombyx mori, which has bombykol as its pheromone component, demonstrated that pheromone titer, body weight, and lipid droplet (LD) diameter in the pheromone gland were affected by the larval diet. Although the artificial diet contained approximately 11-fold more total fatty acids than mulberry leaf, the pheromone titer in the group fed the artificial diet (group AD) was approximately 2-fold higher than that of the group fed mulberry (group M). The diameter of LDs, which store the pheromone-precursor fatty acyl, E10,Z12-16:Acyl, was also larger in the AD group. The relatively small increase in sex pheromone titer by feeding on a fatty-acid-rich diet may be partly attributable to the storage of excess precursors in the LDs. We detected LDs in the pheromone glands of Trilocha varians, the closest non-congener of B. mori available in Bombycidae. T. varians uses bombykal and bombykyl acetate as sex pheromone components, which are biosynthesized via the same precursor fatty acyl as that of B. mori. The presence of LDs in T. varians suggests that the storage and mobilization mechanisms of the pheromone precursor fatty acyl via LDs may be conserved in bombycids.

Niemann-Pick Type C Proteins Are Required for Sterol Transport and Appressorium-Mediated Plant Penetration of Colletotrichum orbiculare.

Many biotrophic and hemibiotrophic fungal pathogens use appressoria to directly penetrate the host plant surface. In the cucumber anthracnose fungus Colletotrichum orbiculare, differentiation of appressoria requires a proper G1/S cell cycle progression, regulated by the GTPase-activating protein complex CoBub2-CoBfa1 and its downstream GTPase CoTem1. To explore the mechanisms by which the CoTem1 cascade regulates plant infection, we screened for CoTem1 interaction factors and identified a Niemann-Pick type C2 homolog (CoNpc2). Niemann-Pick type C proteins NPC1 and NPC2 are sterol-binding proteins required for sterol export from lysosomes (vacuoles) in humans and yeasts. We showed that CoNpc2 colocalized with CoNpc1 in late endosomes and vacuoles and that disruption of its gene resulted in aberrant sterol accumulation in vacuoles and loss of sterol membrane localization, indicating that NPC proteins are engaged in sterol transport in C. orbiculare. For appressorium infection, sterol transport and proper distribution mediated by CoNpc1 and CoNpc2 are critical for membrane integrity and membrane curvature with actin assembly, leading to penetration peg emergence and appressorial cone formation. Our results revealed a novel mechanism by which NPC proteins regulate appressorium-mediated plant infection. IMPORTANCE Fungal morphogenesis requires accurate cell cycle progression. Two-component GTPase-activating protein (GAP) CoBub2-CoBfa1 interacts with downstream GTPase CoTem1 and is required for G1/S progression to establish plant infection in Colletotrichum orbiculare. To understand the pathogenicity related functions of CoTem1 downstream, we identified a Niemann-Pick type C2 homolog (CoNpc2) as a novel physical interaction factor with CoTem1. Whereas NPC proteins (NPC1 and NPC2) are essential for sterol homeostasis in humans and yeasts, their functions in plant invasion by pathogenic fungi have remained unclear. In this study, we show that CoNPC1 and CoNPC2 play a critical role in intracellular sterol transport and that appropriate sterol distribution is required for membrane integrity and membrane curvature with actin assembly that leads to appressorium-mediated plant penetration and pathogenicity of C. orbiculare. Our findings suggest the importance of sterol distribution in fungal morphogenesis during plant infection.

Plant pathogenic fungi Colletotrichum and Magnaporthe share a common G1 phase monitoring strategy for proper appressorium development.

To breach the plant cuticle, many plant pathogenic fungi differentiate specialized infection structures (appressoria). In Colletotrichum orbiculare (cucumber anthracnose fungus), this differentiation requires unique proper G1 /S phase progression, regulated by two-component GTPase activating protein CoBub2/CoBfa1 and GTPase CoTem1. Since their homologues regulate mitotic exit, cytokinesis, or septum formation from yeasts to mammals, we asked whether the BUB2 function in G1 /S progression is specific to plant pathogenic fungi. Colletotrichum higginsianum and Magnaporthe oryzae were genetically analyzed to investigate conservation of BUB2 roles in cell cycle regulation, septum formation, and virulence. Expression profile of cobub2Δ was analyzed using a custom microarray. In bub2 mutants of both fungi, S phase initiation was earlier, and septum formation coordinated with a septation initiation network protein and contractile actin ring was impaired. Earlier G1 /S transition in cobub2Δ results in especially high expression of DNA replication genes and differing regulation of virulence-associated genes that encode proteins such as carbohydrate-active enzymes and small secreted proteins. The virulence of chbub2Δ and mobub2Δ was significantly reduced. Our evidence shows that BUB2 regulation of G1 /S transition and septum formation supports its specific requirement for appressorium development in plant pathogenic fungi.

Colletotrichum orbiculare MTF4 Is a Key Transcription Factor Downstream of MOR Essential for Plant Signal-Dependent Appressorium Development and Pathogenesis.

The cucumber anthracnose fungus Colletotrichum orbiculare forms a specialized infection structure, called an appressorium. Appressorium differentiation relies on fungal perception of physical and biochemical signals at the plant surface. Our previous report showed that the morphogenesis-related NDR (nuclear Dbf2-related) kinase pathway (MOR) is crucial for translating plant-derived signals for appressorium development. Here, we focused on identifying transcriptional regulators downstream of MOR that are involved in plant signal sensing and transduction for appressorium development. Based on whole-genome transcript profiling, we identified a Zn(II)2Cys6 transcription factor, CoMTF4, as a potential downstream factor of MOR. CoMTF4 was expressed in planta rather than in vitro under the control of the NDR kinase CoCbk1. Phenotypes of comtf4 mutants, strains with constitutively active CoCbk1 and strains with constitutive overexpression of CoMTF4 suggested that CoMtf4 acts downstream of MOR. Furthermore, nuclear localization of CoMtf4 was dependent on the MOR and responsive to plant-derived signals that lead to appressorium morphogenesis. Thus, we conclude that CoMtf4 is a transcription factor downstream of MOR that is essential for appressorium morphogenesis and pathogenesis and is regulated in response to plant-derived signals. This study provides insights into fungal sensing of plant signals and subsequent responses critical for appressorium formation.

The morphogenesis-related NDR kinase pathway of Colletotrichum orbiculare is required for translating plant surface signals into infection-related morphogenesis and pathogenesis.

Plant infection by pathogenic fungi involves the differentiation of appressoria, specialized infection structures, initiated by fungal sensing and responding to plant surface signals. How plant fungal pathogens control infection-related morphogenesis in response to plant-derived signals has been unclear. Here we showed that the morphogenesis-related NDR kinase pathway (MOR) of the cucumber anthracnose fungus Colletotrichum orbiculare is crucial for appressorium development following perception of plant-derived signals. By screening of random insertional mutants, we identified that the MOR element CoPag1 (Perish-in-the-absence-of-GYP1) is a key component of the plant-derived signaling pathway involved in appressorium morphogenesis. Constitutive activation of the NDR kinase CoCbk1 (Cell-wall-biosynthesis-kinase-1) complemented copag1 defects. Furthermore, copag1 deletion impaired CoCbk1 phosphorylation, suggesting that CoPag1 functions via CoCbk1 activation. Searching for the plant signals that contribute to appressorium induction via MOR, we found that the cutin monomer n-octadecanal, degraded from the host cuticle by conidial esterases, functions as a signal molecule for appressorium development. Genome-wide transcriptional profiling during appressorium development revealed that MOR is responsible for the expression of a subset of the plant-signal-induced genes with potential roles in pathogenicity. Thus, MOR of C. orbiculare has crucial roles in regulating appressorium development and pathogenesis by communicating with plant-derived signals.