Conserved innate immune system in Pyr. yezoensis
Sensitive PTI mechanism
PTI, triggered by pathogenic PAMPs, are the first line of non-specific defense mechanism exhibited by the plant against the pathogen. In Pyr. yezoensis, after recognition of the carbohydrate PAMPs and lectin RLPs, PTI mechanism is activated in response to Pyt. porphyrae infection. Based on enrichment analysis, several broad-spectrum defense mechanisms could be identified in Pyr. yezoensis, including secondary metabolites, cellulase, metalloproteinase inhibitor, ROS, and HSPs. All these mechanisms were up regulated since the host was exposed to the zoospores at the slightly infected stage. This indicates that the Pyr. yezoensis PTI mechanism is extremely sensitive to infection.
Successful recognition of PAMPs and PRRs was the first step in the activation of innate immunity during infection in the plants . Plant lectins play a crucial role in the recognition and binding of carbohydrate PAMPs. Based on the variability in lectin domains, lectin PRRs could be divided into three subclasses: L-type, G-type, and C-type. L-type lectins bind glucose/mannose (Glc/Man) specifically but the ligand PAMPs of G-type and C-type lectins are still not very evident in the plants. During the infection, Pyr. yezoensis C-type lectin gene was up-regulated only in the slightly infected stage and L-type lectin genes were up-regulated only in the severely infected stage. Thus, suggesting that C-type lectin might recognize PAMPs expressed by Pyt. porphyrae and then modulate defense gene expression before direct invasion of the pathogen. L-type lectin might activate or amplify the PTI in severely infected stage by recognizing glucose and mannose released due to the degradation of the Pyr. yezoensis cell wall that consists of the mannan outer layer and xylan microfibrils in the inner layer [43, 44].
It has been reported that several secondary metabolites in red algae Laurencia majuscule exhibit antimicrobial activity against “ice-ice” bacteria . In crops, secondary metabolites were frequently used to nondestructively detect diseases using MCFI [46, 47]. In the present study, MCFI was first used to measure the concentration of secondary metabolites in diseased algae. The results of F520 indicates Pyr. yezoensis induces secondary metabolite expression before direct invasion of the hyphae, suggesting that Pyr. yezoensis secondary metabolism is sensitive to Pyt. porphyrae infection and might be used in the nondestructive detection of red rot disease.
Protease inhibitors (PIs), a part of plant ETI, protect the host by inhibiting pathogen proteases and regulating the activity of host protease. It has been proved that PIs from Hordeum vulgare, Vicia faba, and A. thaliana could inhibit the mycelial growth of several fungal pathogens (broad-spectrum inhibition)[49-51]. In the present study, an extracellular metalloproteinase inhibitor is identified for the first time in the algal innate immune system. Consistent with the transcriptomic and proteomic data this protein expression was up-regulated significantly (FC=1.52) after infection (further analysis of data in ). It appears that Pyr. yezoensis secretes metalloproteinase inhibitor as a “bait” to occupy the metalloproteinase catalytic center and protects the host protein degradation by pathogen metalloproteinase.
Plants can inhibit the fungal pathogens by secreting chitinase and β-1, 3-glucanase that degrade fungal cell wall during infection[52, 53]. In Pyr. tenera, several cell-wall associated hydrolases were up-regulated after Pyt. Porphyrae invasion. In the current study, we analyzed the pathogen cell wall degrading enzymes in detail. The main constituent of the oomycete cell wall is β-1,3-, β-1,4-, and β-1,6-linked glucan skeleton with cellulose and is different from the fungal cell wall. The Pyr. yezoensis cellulase belongs to glycosyl hydrolase family 5 (cellulase) and was continuously up-regulated during the whole infection process. It might be possible that Pyr. yezoensis secreted cellulase to degrade cellulose, thereby inhibiting the formation and expansion of Pyt. porphyrae hyphae.
Large HSPs and single R-protein domain-containing proteins act as R-proteins in Pyr. yezoensis
R proteins are essential for the plant to activate ETI and create specificity in disease resistance. R proteins with conserved protein structure are extensively present in higher plants. The increase in R-protein expression indicates the plausibility of these genes to be involved in Pyr. yezoensis innate immunity. However, owing to their simple protein structures, the function of these genes might be limited in the interaction of Pyr. yezoensis and Pyt. porphyrae.
Several findings demonstrated that large HSPs, such as HSP70s and HSP90s, could recognize pathogen effectors specifically and activate the defense mechanisms against pathogens . Results from yeast two-hybrid analysis and co-immunoprecipitation indicate that HSP90s might play a role similar to R proteins in plants [55, 56]. Considering the absence of NBS-LRR proteins, the up-regulated HSP70s and HSP90s might serve as substitutes for a typical R protein in ETI activation of Pyr. yezoensis innate immune system.
Conversed ETI in Pyr. yezoensis
Hypersensitive response (HR), a form of programmed cell death, gets triggered after recognition of R protein and the effectors at the site of infection to prevent the spread of the pathogen. It has been established that metacaspases are the key factors in plant PCD during infection. AtMC1 and AtMC2, two metacaspases in Arabidopsis, possess opposing roles in PCD during Pseudomonas syringae infection. However, little is known about the mechanism of PCD in algae during biotic stress. Wang et al., hypothesized that flg22, a typical bacterial PAMP, could induce PCD in female gametophyte of S. japonica depending on the cell ultrastructural changes during infection. It is well known that as an ETI mechanism, PCD is more efficient in resisting biotrophic pathogens but is limited in the defense against necrotrophic pathogens. As necrotrophic pathogens, Pyt. porphyrae could feed on the dead cells or tissues. Nevertheless, PCD related genes that were up-regulated include metacaspase, endonuclease G, and cytochrome C, and these still could be identified in Pyr. yezoensis transcripts during infection. These results indicate that PCD is involved in the immune response of Pyr. yezoensis against Pyt. porphyrae.
In plants, UPS is associated with innate immunity mechanism in a variety of ways, such as PTI modulation, programmed cell death, and signal transduction. E3 is the key enzyme in the process of ubiquitination as it binds to the target protein specifically via its target recognition subunit[60, 61]. In Pyr. yezoensis, genes related to UPS were up-regulated during infection. Especially in severely infected stage, all parts of UPS were up-regulated significantly, indicating that UPS might play a crucial role in Pyr. yezoensis to defend the invasion of Pyt. porphyrae hyphae in the severely infected stage.
The present study reasonably deduced the overall view of Pyr. yezoensis innate immunity (Fig. 6). During the slightly infected stage, Pyr. yezoensis cellulase is induced and secreted to degrade Pyt. porphyrae cell wall. Meanwhile, several types of oligosaccharides are produced that might be recognized as carbohydrate PAMPs by C-type lectins on the host membrane, these work in concert with an unknown kinase to activate the PTI mechanism. NADPH oxidase is induced to generate ROS. Meanwhile, antioxidase also is up-regulated to overcome oxidative stress. Secondary metabolism, especially the synthesis of phenolics is induced. A metalloproteinase inhibitor was shown to be highly induced and secreted to inhibit pathogen metalloproteinase activity. Several HSP20s, which are involved in damage repair, also are highly up-regulated. In severely infected stage, hyphae invade into cells of Pyr. yezoensis. Mannose gets released during the cell wall degradation of Pyr. yezoensis. Host L-type lectin recognizes the mannose to further activate and amplify the PTI mechanism. Besides, Pyr. yezoensis may rely on ETI to prevent the invasion of a pathogen. In Pyr. yezoensis, ETI gets activated by potential R-proteins containing typical R-protein domain. Hsp70s and HSP90s might play the role of R-proteins to interact with pathogen effectors. Similar to that seen in higher plants, in Pyr. yezoensis ETI, mechanisms described in the PTI were amplified. The ubiquitin system and PCD also get up-regulated to resist the infection.
The ancient origin of genes in the innate immune system of Pyr. yezoensis
Endosymbiosis drives the evolution and diversification of eukaryotic algae. Plastids originated from cyanobacteria and created three “primary algae”: Glaucophyta, Rhodophyta, and Chlorophyta. Chlorophyta further evolved to land plants. The current study revealed that the defense mechanism in Pyr. yezoensis is similar to that seen in plants. Nevertheless, phylogenetic analysis revealed that the defense genes in Pyr. yezoensis might have an ancient origin.
PRRs are transmembrane proteins with functional ectodomains binding to PAMPs . Based on whether the kinase domain is intracellular in origin or not, PRRs are classified into receptor-like kinases (RLKs) and receptor-like proteins (RLPs) in plants. So far, all the identified lectin PRRs in plants belong to RLKs. However, due to the absence of intracellular kinase domain, lectins in Pyr. yezoensis belongs to RLPs. Phylogenetic analysis revealed that the Pyr. yezoensis lectin preserved ancient protein structure and originated earlier than plant lectins (Fig S2-S3). It has been reported that plant RLPs need an independent kinase to work together . For instance, in tomato, Avr9/Cf‐9 induced kinase 1 (ACIK1) is required to interact with LRR-RLP Cf-9 through the assistance of Cf‐9 interacting thioredoxin (CITRX) as a chaperone. Therefore, we surmised that an unknown kinase is required to work with Pyr. yezoensis lectin to activate the downstream defense genes.
Nearly all the identified R proteins typically share commonly conversed domains of TNL (TIR-NBS- LRR) or nTNL (NBS- LRR). Recent studies analyzed the R protein in plants, Charophytes, Chlorophyta, Rhodophyta, and Glaucophyta[66, 67]. Proteins containing NBS or LRR domain are found to exist in all species. However, the fusion events of NBS and LRR domain was found in Chlorophyta and plants. This indicated that the origin of R protein could be traced back to Chlorophyta, such as Chromochloris zofingiensis and Botryococcus braunii . In the current study, six up-regulated genes encoding NBS, LRR, or TIR containing proteins were predicted as R protein candidates in Pyr. yezoensis. As shown in Figure S4, these genes originate anciently compared to typical R proteins in plants. Structural analysis showed that all these genes encode an ancient protein structure of a single R protein domain without fusion events. The phenomenon of gene fusion was generally found during the evolution of organisms. In archaea and bacteria, the glutamyl‐ and prolyl‐tRNA synthetases (GluRS and ProRS) are encoded by two distinct genes. However, a single polypeptide chain protein combining GluRS and ProRS were present in the eukaryotic phylum of coelomate metazoans. Similar co-working mechanism also exists in Arabidopsis R proteins. Due to the lack of LRR, TIR-NBS and TIR-X proteins could interact with NBS-LRR. This suggests that proteins containing a single R protein domain could also act as R proteins with the assistance of other proteins. Therefore, we assumed that single domain containing protein (NBS, TIR, or LRR) might play the role of R protein by working in concert.
As shown in Figure S5–S7, the PTI genes of Pyr. yezoensis, that include cellulase, metalloproteinase inhibitor, and NADH-oxidase were clustered into a clade of Rhodophyta, which is separate from the clade of plants and Chlorophyta. Therefore, our findings indicate that Pyr. yezoensis defense genes preserve the original character in the evolutionary process of plant innate immune system.