Emerging Roles of Motile Epidermal Chloroplasts in Plant Immunity.

Plant epidermis contains atypical small chloroplasts. However, the physiological role of this organelle is unclear compared to that of large mesophyll chloroplasts, the well-known function of which is photosynthesis. Although knowledge of the involvement of chloroplasts in the plant immunity has been expanded to date, the differences between the epidermal and mesophyll chloroplasts are beyond the scope of this study. Given the role of the plant epidermis as a barrier to environmental stresses, including pathogen attacks, and the immune-related function of chloroplasts, plant defense research on epidermal chloroplasts is an emerging field. Recent studies have revealed the dynamic movements of epidermal chloroplasts in response to fungal and oomycete pathogens. Furthermore, epidermal chloroplast-associated proteins and cellular events that are tightly linked to epidermal resistance against pathogens have been reported. In this review, I have focused on the recent progress in epidermal chloroplast-mediated plant immunity.

Preinvasive nonhost resistance of Arabidopsis against melanized appressorium-mediated entry of multiple nonadapted Colletotrichum fungi.

Nonhost plants effectively block a vast number of nonadapted fungal pathogens at the preinvasive stage. On the host plants, adapted fungal pathogens such as Colletotrichum species invade into plant epidermal cell by penetration peg developed from melanized appressorium, followed by invasive hyphal extension. I reported nonadapted Colletotrichum fungi that showed an increased rate of melanized appressorium-mediated entry (MAE) into the pen2 mutant of nonhost Arabidopsis thaliana (hereafter Arabidopsis). It was also found that other MAE-type nonadapted Colletotrichum fungi with no penetration into the pen2 mutant invaded Arabidopsis in the presence of additional mutations such as edr1, gsh1, eds5, cas, and chup1 in the pen2 background. Thus, many immune components contribute to the preinvasive nonhost resistance (NHR) of Arabidopsis against Colletotrichum MAE, and PEN2-related defense takes priority over other defense pathways. Here, I show that among the above nonadapted fungi, Colletotrichum nymphaeae PL1-1-b exhibited relatively lower incompatibility with the nonhost Arabidopsis with increased MAE in each single mutant of edr1, gsh1, eds5, and cas, although other nonadapted fungi almost never invaded these single mutants. Based on the relationships between Colletotrichum MAE and the Arabidopsis immune-related components, Colletotrichum-Arabidopsis incompatibility and multilayered immunity in the preinvasive NHR of Arabidopsis are discussed in this study.

Conserved fungal effector suppresses PAMP-triggered immunity by targeting plant immune kinases.

Plant pathogens have optimized their own effector sets to adapt to their hosts. However, certain effectors, regarded as core effectors, are conserved among various pathogens, and may therefore play an important and common role in pathogen virulence. We report here that the widely distributed fungal effector NIS1 targets host immune components that transmit signaling from pattern recognition receptors (PRRs) in plants. NIS1 from two Colletotrichum spp. suppressed the hypersensitive response and oxidative burst, both of which are induced by pathogen-derived molecules, in Nicotiana benthamianaMagnaporthe oryzae NIS1 also suppressed the two defense responses, although this pathogen likely acquired the NIS1 gene via horizontal transfer from Basidiomycota. Interestingly, the root endophyte Colletotrichum tofieldiae also possesses a NIS1 homolog that can suppress the oxidative burst in N. benthamiana We show that NIS1 of multiple pathogens commonly interacts with the PRR-associated kinases BAK1 and BIK1, thereby inhibiting their kinase activities and the BIK1-NADPH oxidase interaction. Furthermore, mutations in the NIS1-targeting proteins, i.e., BAK1 and BIK1, in Arabidopsis thaliana also resulted in reduced immunity to Colletotrichum fungi. Finally, M. oryzae lacking NIS1 displayed significantly reduced virulence on rice and barley, its hosts. Our study therefore reveals that a broad range of filamentous fungi maintain and utilize the core effector NIS1 to establish infection in their host plants and perhaps also beneficial interactions, by targeting conserved and central PRR-associated kinases that are also known to be targeted by bacterial effectors.

Focal effector accumulation in a biotrophic interface at the primary invasion sites of Colletotrichum orbiculare in multiple susceptible plants.

We identified virulence-related effectors of a hemibiotrophic fungal pathogen Colletotrichum orbiculare, and found that a novel interface was generated by a biotrophic interaction between C. orbiculare and the host cucumber, in which the effectors secreted from the pathogen accumulated preferentially. The interface was located around the biotrophic primary hyphal neck. Here, we showed that C. orbiculare also developed this interface in a biotrophic interaction with melon, which belongs to Cucurbitaceae. Furthermore, C. orbiculare developed interface in the interaction with a susceptible plant, Nicotiana benthamiana, which is distantly related to Cucurbitaceae, suggesting that the spatial regulation strategy for effectors in C. orbiculare is not specific to cucumber; rather, it is conserved among the various plants that are susceptible to this pathogen.

Comparative genomic and transcriptomic analyses reveal the hemibiotrophic stage shift of Colletotrichum fungi.

Hemibiotrophic fungal plant pathogens represent a group of agronomically significant disease-causing agents that grow first on living tissue and then cause host death in later, necrotrophic growth. Among these, Colletotrichum spp. are devastating pathogens of many crops. Identifying expanded classes of genes in the genomes of phytopathogenic Colletotrichum, especially those associated with specific stages of hemibiotrophy, can provide insights on how these pathogens infect a large number of hosts. The genomes of Colletotrichum orbiculare, which infects cucurbits and Nicotiana benthamiana, and C. gloeosporioides, which infects a wide range of crops, were sequenced and analyzed, focusing on features with potential roles in pathogenicity. Regulation of C. orbiculare gene expression was investigated during infection of N. benthamiana using a custom microarray. Genes expanded in both genomes compared to other fungi included sequences encoding small, secreted proteins (SSPs), secondary metabolite synthesis genes, proteases and carbohydrate-degrading enzymes. Many SSP and secondary metabolite synthesis genes were upregulated during initial stages of host colonization, whereas the necrotrophic stage of growth is characterized by upregulation of sequences encoding degradative enzymes. Hemibiotrophy in C. orbiculare is characterized by distinct stage-specific gene expression profiles of expanded classes of potential pathogenicity genes.

Large-scale gene disruption in Magnaporthe oryzae identifies MC69, a secreted protein required for infection by monocot and dicot fungal pathogens.

To search for virulence effector genes of the rice blast fungus, Magnaporthe oryzae, we carried out a large-scale targeted disruption of genes for 78 putative secreted proteins that are expressed during the early stages of infection of M. oryzae. Disruption of the majority of genes did not affect growth, conidiation, or pathogenicity of M. oryzae. One exception was the gene MC69. The mc69 mutant showed a severe reduction in blast symptoms on rice and barley, indicating the importance of MC69 for pathogenicity of M. oryzae. The mc69 mutant did not exhibit changes in saprophytic growth and conidiation. Microscopic analysis of infection behavior in the mc69 mutant revealed that MC69 is dispensable for appressorium formation. However, mc69 mutant failed to develop invasive hyphae after appressorium formation in rice leaf sheath, indicating a critical role of MC69 in interaction with host plants. MC69 encodes a hypothetical 54 amino acids protein with a signal peptide. Live-cell imaging suggested that fluorescently labeled MC69 was not translocated into rice cytoplasm. Site-directed mutagenesis of two conserved cysteine residues (Cys36 and Cys46) in the mature MC69 impaired function of MC69 without affecting its secretion, suggesting the importance of the disulfide bond in MC69 pathogenicity function. Furthermore, deletion of the MC69 orthologous gene reduced pathogenicity of the cucumber anthracnose fungus Colletotrichum orbiculare on both cucumber and Nicotiana benthamiana leaves. We conclude that MC69 is a secreted pathogenicity protein commonly required for infection of two different plant pathogenic fungi, M. oryzae and C. orbiculare pathogenic on monocot and dicot plants, respectively.

Cell death of Nicotiana benthamiana is induced by secreted protein NIS1 of Colletotrichum orbiculare and is suppressed by a homologue of CgDN3.

Colletotrichum orbiculare, the causal agent of cucumber anthracnose, infects Nicotiana benthamiana. Functional screening of C. orbiculare cDNAs in a virus vector-based plant expression system identified a novel secreted protein gene, NIS1, whose product induces cell death in N. benthamiana. Putative homologues of NIS1 are present in selected members of fungi belonging to class Sordariomycetes, Dothideomycetes, or Orbiliomycetes. Green fluorescent protein-based expression studies suggested that NIS1 is preferentially expressed in biotrophic invasive hyphae. NIS1 lacking signal peptide did not induce NIS1-triggered cell death (NCD), suggesting apoplastic recognition of NIS1. NCD was prevented by virus-induced gene silencing of SGT1 and HSP90, indicating the dependency of NCD on SGT1 and HSP90. Deletion of NIS1 had little effect on the virulence of C. orbiculare against N. benthamiana, suggesting possible suppression of NCD by C. orbiculare at the postinvasive stage. The CgDN3 gene of C. gloeosporioides was previously identified as a secreted protein gene involved in suppression of hypersensitive-like response in Stylosanthes guianensis. Notably, we found that NCD was suppressed by the expression of a CgDN3 homologue of C. orbiculare. Our findings indicate that C. orbiculare expresses NIS1 at the postinvasive stage and suggest that NCD could be repressed via other effectors, including the CgDN3 homologue.

Control of chemotactic signal gain via modulation of a pre-formed receptor array.

The remarkably wide dynamic range of the chemotactic pathway of Escherichia coli, a model signal transduction system, is achieved by methylation/amidation of the transmembrane chemoreceptors that regulate the histidine kinase CheA in response to extracellular stimuli. The chemoreceptors cluster at a cell pole together with CheA and the adaptor CheW. Several lines of evidence have led to models that assume high cooperativity and sensitivity via collaboration of receptor dimers within a cluster. Here, using in vivo disulfide cross-linking assays, we have demonstrated a well defined arrangement of the aspartate chemoreceptor (Tar). The differential effects of amidation on cross-linking at different positions indicate that amidation alters the relative orientation of Tar dimers to each other (presumably inducing rotational displacements) without much affecting the conformation of the periplasmic domains. Interestingly, the effect of aspartate on cross-linking at any position tested was roughly opposite to that of receptor amidation. Furthermore, amidation attenuated the effects of aspartate by several orders of magnitude. These results suggest that receptor covalent modification controls signal gain by altering the arrangement or packing of receptor dimers in a pre-formed cluster.