Search

The hemibiotrophic fungal plant pathogen Colletotrichum orbiculare is predicted to secrete hundreds of effector proteins when the pathogen infects cucurbit crops, such as cucumber and melon, and tobacco (Nicotiana benthamiana), a distantly related Solanaceae species. Here, we report the identification of sets of C. orbiculare effector genes that are differentially required for fungal virulence to two phylogenetically distant host species. Through targeted gene knockout screening of C. orbiculare ‘core’ effector candidates defined based on in planta gene expression, we identified: four host-specific virulence effectors (named effector proteins for cucurbit infection, or EPCs) that are required for full virulence of C. orbiculare to cucurbit hosts, but not to the Solanaceae host N. benthamiana; and five host-nonspecific virulence effectors, which collectively contribute to fungal virulence to both hosts. During host infection, only a small subset of genes, including the host-specific EPC effector genes, showed preferential expression on one of the hosts, while gene expression profiles of the majority of other genes, including the five host-nonspecific effector genes, were common to both hosts. This work suggests that C. orbiculare adopts a host-specific effector deployment strategy, in addition to general host-blind virulence mechanisms, for adaptation to cucurbit hosts.

Microbial pathogens strategically acquire metabolites from their hosts during infection. Here we show that the host can intervene to prevent such metabolite loss to pathogens. Phosphorylation-dependent regulation of sugar transporter 13 (STP13) is required for antibacterial defense in the plant Arabidopsis thaliana. STP13 physically associates with the flagellin receptor flagellin-sensitive 2 (FLS2) and its co-receptor BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 (BAK1). BAK1 phosphorylates STP13 at threonine 485, which enhances its monosaccharide uptake activity to compete with bacteria for extracellular sugars. Limiting the availability of extracellular sugar deprives bacteria of an energy source and restricts virulence factor delivery. Our results reveal that control of sugar uptake, managed by regulation of a host sugar transporter, is a defense strategy deployed against microbial infection. Competition for sugar thus shapes host-pathogen interactions.

Effective defense of Arabidopsis against filamentous pathogens requires two mechanisms, both of which involve biosynthesis of tryptophan (Trp)-derived metabolites. Extracellular resistance involves products of PEN2-dependent metabolism of indole glucosinolates (IGs). Restriction of further fungal growth requires PAD3-dependent camalexin and other, as yet uncharacterized, indolics. This study focuses on the function of CYP71A12 monooxygenase in pathogen-triggered Trp metabolism, including the biosynthesis of indole-3-carboxylic acid (ICA). Moreover, to investigate the contribution of CYP71A12 and its products to Arabidopsis immunity, we analyzed infection phenotypes of multiple mutant lines combining pen2 with pad3, cyp71A12, cyp71A13 or cyp82C2. Metabolite profiling of cyp71A12 lines revealed a reduction in ICA accumulation. Additionally, analysis of mutant plants showed that low amounts of ICA can form during an immune response by CYP71B6/AAO1-dependent metabolism of indole acetonitrile, but not via IG hydrolysis. Infection assays with Plectosphaerella cucumerina and Colletotrichum tropicale, two pathogens with different lifestyles, revealed cyp71A12-, cyp71A13- and cyp82C2-associated defects associated with Arabidopsis immunity. Our results indicate that CYP71A12, but not CYP71A13, is the major enzyme responsible for the accumulation of ICA in Arabidopsis in response to pathogen ingression. We also show that both enzymes are key players in the resistance of Arabidopsis against selected filamentous pathogens after they invade.

New evidences are emerging to support the importance of viral replication complexes (VRCs) in not only viral replication, but also viral cell-to-cell movement. Currently, how VRCs grow in size and colocalize with viral movement proteins (MPs) remains unclear. Herein, we performed live-cell imaging of red clover necrotic mosaic virus (RCNMV) dsRNA by using reporter B2-GFP plants. Tiny granules of dsRNA were formed along the endoplasmic reticulum (ER) at an early stage of infection. Importantly, the colocalization of the dsRNA granules with the virus-encoded p27 replication protein showed that these structures are components of VRCs. These granules moved throughout the cytoplasm, driven by the acto–myosin system, and coalesced with each other to form larger aggregates; the MPs were not associated with these processes. Notably, the MPs colocalized preferentially with large dsRNA aggregates, rather than with tiny dsRNA granules, suggesting that the increase in the size of VRCs promotes their colocalization with MPs.

In addition to conspicuous large mesophyll chloroplasts, where most photosynthesis occurs, small epidermal chloroplasts have also been observed in plant leaves. However, the functional significance of this small organelle remains unclear. Here, we present evidence that Arabidopsis epidermal chloroplasts control the entry of fungal pathogens. In entry trials, specialized fungal cells called appressoria triggered dynamic movement of epidermal chloroplasts. This movement is controlled by common regulators of mesophyll chloroplast photorelocation movement, designated as the epidermal chloroplast response (ECR). The ECR occurs when the PEN2 myrosinase-related higher-layer antifungal system becomes ineffective, and blockage of the distinct steps of the ECR commonly decreases preinvasive nonhost resistance against fungi. Furthermore, immune components were preferentially localized to epidermal chloroplasts, contributing to antifungal nonhost resistance in the pen2 background. Our findings reveal that atypical small chloroplasts act as defense-related motile organelles by specifically positioning immune components in the plant epidermis, which is the first site of contact between the plant and pathogens. Thus, this work deepens our understanding of the functions of epidermal chloroplasts.

Colletotrichum orbiculare infects cucurbits, such as cucumber and melon (Cucumis melo), as well as the model Solanaceae plant Nicotiana benthamiana, by secreting an arsenal of effectors that suppress the immunity of these distinct plants. Two conserved effectors of C. orbiculare, called NLP1 and NIS1, induce cell death responses in N. benthamiana, but it is unclear whether they exhibit the same activity in Cucurbitaceae plants. In this study, we established a new Agrobacterium-mediated transient expression system to investigate the cell death-inducing activity of NLP1 and NIS1 in melon. NLP1 strongly induced cell death in melon but, in contrast to the effects seen in N. benthamiana, mutations either in the heptapeptide motif or in the putative glycosylinositol phosphorylceramide-binding site did not cancel its cell death-inducing activity in melon. Furthermore, NLP1 lacking the signal peptide caused cell death in melon but not in N. benthamiana. Study of the transient expression of NIS1 also revealed that, unlike in N. benthamiana, NIS1 did not induce cell death in melon. In contrast, NIS1 suppressed flg22-induced reactive oxygen species generation in melon, as seen in N. benthamiana. These findings indicate distinct cell death-inducing activities of NLP1 and NIS1 in these two plant species that C. orbiculare infects.

Plants recognize pathogen-associated molecular patterns (PAMPs) to activate PAMP-triggered immunity (PTI). However, our knowledge of PTI signaling remains limited. In this report, we introduce Lumi-Map, a high-throughput platform for identifying causative single nucleotide polymorphisms (SNPs) to studying PTI signaling components. In Lumi-Map, a transgenic reporter plant line is produced that contains a firefly luciferase (LUC) gene driven by a defense gene promoter, which generates luminescence upon PAMP treatment. The line is mutagenized and the mutants with altered luminescence patterns are screened by a high-throughput real-time bioluminescence monitoring system. Selected mutants are subjected to MutMap analysis, a whole genome sequencing (WGS)-based method of rapid mutation identification, to identify the causative SNP responsible for the luminescence pattern change. We generated nine transgenic Arabidopsis reporter lines expressing LUC gene fused to multiple promoter sequences of defense-related genes. These lines generate luminescence upon activation of FLAGELLIN-SENSING 2 (FLS2) by flg22, a PAMP derived from bacterial flagellin. We selected the WRKY29-promoter reporter line to identify mutants in the signaling pathway downstream of FLS2. After screening 24,000 ethylmethanesulfonate (EMS)-induced mutants of the reporter line, we isolated 22 mutants with altered WRKY29 expression upon flg22 treatment (abbreviated as awf mutants). While five flg22-insensitive awf mutants harbored mutations in FLS2 itself, Lumi-Map revealed three genes not previously associated with PTI. Lumi-Map has the potential to identify novel PAMPs and their receptors as well as signaling components downstream of the receptors.