Plant-biotic interaction
MicroRNAs (miRNAs) play important roles in rice response to Magnaporthe oryzae, the causative agent of rice blast disease. Studying the roles of rice miRNAs is of great significance for the disease control. Osa‐miR167d belongs to a conserved miRNA family targeting auxin responsive factor (ARF) genes that act in developmental and stress‐induced responses. Here, we show that Osa‐miR167d plays a negative role in rice immunity against M. oryzae by suppressing its target gene. The expression of Osa‐miR167d was significantly suppressed in a resistant accession at and after 24 h post inoculation (hpi), however, its expression was significantly increased at 24 hpi in the susceptible accession upon M. oryzae infection. Transgenic rice lines over‐expressing Osa‐miR167d were highly susceptible to multiple blast fungal strains. By contrast, transgenic lines expressing a target mimicry to block Osa‐miR167d enhanced resistance to rice blast disease. In addition, knocking out the target gene ARF12 led to hyper‐susceptibility to multiple blast fungal strains. Taken together, our results indicate that Osa‐miR167d negatively regulate rice immunity to facilitate the infection of M. oryzae by downregulating ARF12. Thus, Osa‐miR167d‐ARF12 regulatory module could be valuable in improvement of blast‐disease resistance.
Plants have evolved multiple defense strategies to cope with pathogens, among which plant immune signaling that relies on cell‐surface localized and intracellular receptors takes fundamental roles. Exciting breakthroughs were made recently on the signaling mechanisms of pattern recognition receptors (PRRs) and intracellular nucleotide‐binding site (NBS) and leucine‐rich repeat (LRR) domain receptors (NLRs). This review summarizes the current view of PRRs activation, emphasizing the most recent discoveries about PRRs’ dynamic regulation and signaling mechanisms directly leading to downstream molecular events including mitogen‐activated protein kinase (MAPK) activation and calcium (Ca2+) burst. Plants also have evolved intracellular NLRs to perceive the presence of specific pathogen effectors and trigger more robust immune responses. We also discuss the current understanding of the mechanisms of NLR activation, which has been greatly advanced by recent breakthroughs including structures of the first full‐length plant NLR complex, findings of NLR sensor‐helper pairs and novel biochemical activity of Toll/interleukin‐1 receptor (TIR) domain.
Despite the recent discovery that trehalose synthesis is important for plant development and abiotic stress tolerance, the effects of trehalose on biotic stress responses remain relatively unknown. In this study, we demonstrate that TREHALOSE PHOSPHATE SYNTHASE 5 (TPS5)-dependent trehalose metabolism regulates Arabidopsis thaliana defenses against pathogens (necrotrophic Botrytis cinerea and biotrophic Pseudomonas syringae). Pathogen infection increased trehalose levels and upregulated TPS5 expression. Application of exogenous trehalose significantly improved plant defenses against B. cinerea, but increased the susceptibility of plants to P. syringae. We demonstrate that elevated trehalose biosynthesis, in transgenic plants over-expressing TPS5, also increased the susceptibility to P. syringae, but decreased the disease symptoms caused by B. cinerea. The knockout of TPS5 prevented the accumulation of trehalose and enhanced defense responses against P. syringae. Additionally, we observed that a TPS5-interacting protein (multiprotein bridging factor 1c) was required for induced expression of TPS5 during pathogen infections. Furthermore, we show that trehalose promotes P. syringae growth and disease development, via a mechanism involving suppression of the plant defense gene, Pathogenesis-Related Protein 1. These findings provide insight into the function of TPS5-dependent trehalose metabolism in plant basal defense responses.
Secondary plant metabolites, represented by indole glucosinolates (IGS) and camalexin, play important roles in Arabidopsis immunity. Previously, we demonstrated the importance of MPK3 and MPK6, two closely related MAPKs, in regulating Botrytis cinerea (Bc)‐induced IGS and camalexin biosynthesis. Here we report that CPK5 and CPK6, two redundant calcium‐dependent protein kinases (CPKs), are also involved in regulating the biosynthesis of these secondary metabolites. The loss‐of‐function of both CPK5 and CPK6 compromises plant resistance to Bc. Expression profiling of CPK5‐VK transgenic plants, in which a truncated constitutively active CPK5 is driven by a steroid‐inducible promoter, revealed that biosynthetic genes of both IGS and camalexin pathways are coordinately upregulated after the induction of CPK5‐VK, leading to high‐level accumulation of camalexin and 4‐methoxyindole‐3‐yl‐methylglucosinolate (4MI3G). Induction of camalexin and 4MI3G, as well as the genes in their biosynthesis pathways, is greatly compromised in cpk5 cpk6 mutant in response to Bc. In a conditional cpk5 cpk6 mpk3 mpk6 quadruple mutant, Bc resistance and induction of IGS and camalexin are further reduced in comparison to either cpk5 cpk6 or conditional mpk3 mpk6 double mutant, suggesting that both CPK5/CPK6 and MPK3/MPK6 signaling pathways contribute to promote the biosynthesis of 4MI3G and camalexin in defense against Bc.
The oomycete pathogen Phytophthora sojae is a causal agent of soybean root rot. Upon colonization of soybeans, P. sojae secretes various RXLR effectors to suppress host immune responses, supporting successful infection. Previous research has demonstrated that the RXLR effector Avh94 functions as a virulence effector, but the molecular mechanism underlying its role in virulence remains unknown. Here, we demonstrate that Avh94 overexpression in plants and pathogens promotes Phytophthora infection. Avh94 interacts with soybean JAZ1/2, which is a repressor of jasmonic acid (JA) signaling. Avh94 stabilizes JAZ1/2 to inhibit JA signaling and silencing of JAZ1/2 enhances soybean resistance against P. sojae. Moreover, P. sojae lines overexpressing Avh94 inhibit JA signaling. Furthermore, exogenous application of methyl jasmonate improves plant resistance to Phytophthora. Taken together, these findings suggest that P. sojae employs an RXLR effector to hijack JA signaling and thereby promote infection.
Isochorismate synthase (ICS) plays an essential role in the accumulation of salicylic acid (SA) and plant disease resistance. Diseases caused by Botryosphaeria dothidea affect apple yields. Thus, it is important to understand the role of ICS1 in disease resistance to B. dothidea in apple. In this study, SA treatment enhanced the resistance to B. dothidea. MdICS1 was induced by B. dothidea and enhanced the resistance to B. dothidea. MdICS1 promoter analysis indicated that the W‐box was vital for the response to B. dothidea treatment. MdWRKY15 was found to interact with the W‐box using yeast one‐hybrid screening. Subsequently, the interaction was confirmed by EMSA, yeast one‐hybrid, ChIP‐PCR, and quantitative PCR assays. Moreover, luciferase and GUS analysis further indicated that MdICS1 was transcriptionally activated by MdWRKY15. Finally, we found the function of MdWRKY15 in the resistance to B. dothidea was partially dependent on MdICS1 from the phenotype of transgenic apples and calli. In summary, we revealed that MdWRKY15 activated the transcription of MdICS1 by directly binding to its promoter to increase the accumulation of SA and the expression of disease‐related genes, thereby resulting in the enhanced resistance to B. dothidea in the SA biosynthesis pathway.
Jasmonic acid (JA) plays a critical role in plant defenses against insects and necrotrophic fungi. Wounding or lepidopteran insect feeding rapidly induces a burst of JA in plants, which usually reaches peak values within 1 to 2 h. The induced JA is converted to JA‐Ile and perceived by the COI1‐JAZ co‐receptor, leading to activation of the transcription factors MYC2 and its homologs, which further induce JA‐responsive genes. Although much is known about JA biosynthesis and catabolism enzymes and JA signaling, how JA biosynthesis and catabolism are regulated remain unclear. Here, we show that in Arabidopsis thaliana MYC2 functions additively with MYC3 and MYC4 to regulate wounding‐induced JA accumulation by directly binding to the promoters of genes function in JA biosynthesis and catabolism to promote their transcription. MYC2 also controls the transcription of JAV1 and JAM1 , which are key factors controlling JA biosynthesis and catabolism, respectively. In addition, we also found that MYC2 could bind to the MYC2 promoter and self‐inhibit its own expression. This work illustrates the central role of MYC2/3/4 in controlling wounding‐induced JA accumulation by regulating the transcription of genes involved in JA biosynthesis and catabolism.
Arabidopsis MITOGEN-ACTIVATED PROTEIN KINASE3 (MAPK3 or MPK3) and MPK6 play important signaling roles in plant immunity and growth/development. MAPK KINASE4 (MKK4) and MKK5 function redundantly upstream of MPK3 and MPK6 in these processes. YODA (YDA), also known as MAPK KINASE KINASE4 (MAPKKK4), is upstream of MKK4/MKK5 and forms a complete MAPK cascade (YDA–MKK4/MKK5–MPK3/MPK6) in regulating plant growth and development. In plant immunity, MAPKKK3 and MAPKKK5 function redundantly upstream of the same MKK4/MKK5–MPK3/MPK6 module. However, the residual activation of MPK3/MPK6 in the mapkkk3 mapkkk5 double mutant in response to flg22 pathogen-associated molecular pattern (PAMP) treatment suggests the presence of additional MAPKKK(s) in this MAPK cascade in signaling plant immunity. To investigate whether YDA is also involved in plant immunity, we attempted to generate mapkkk3 mapkkk5 yda triple mutants. However, it was not possible to recover one of the double mutant combinations (mapkkk5 yda) or the triple mutant (mapkkk3 mapkkk5 yda) due to a failure of embryogenesis. Using the clustered regularly interspaced short palindromic repeats (CRISPR) – CRISPR-associated protein 9 (Cas9) approach, we generated weak, N-terminal deletion alleles of YDA, yda-del, in a mapkkk3 mapkkk5 background. PAMP-triggered MPK3/MPK6 activation was further reduced in the mapkkk3 mapkkk5 yda-del mutant, and the triple mutant was more susceptible to pathogen infection, suggesting YDA also plays an important role in plant immune signaling. In addition, MAPKKK5 and, to a lesser extent, MAPKKK3 were found to contribute to gamete function and embryogenesis, together with YDA. While the double homozygous mapkkk3 yda mutant showed the same growth and development defects as the yda single mutant, mapkkk5 yda double mutant and mapkkk3 mapkkk5 yda triple mutants were embryo lethal, similar to the mpk3 mpk6 double mutants. These results demonstrate that YDA, MAPKKK3, and MAPKKK5 have overlapping functions upstream of the MKK4/MKK5–MPK3/MPK6 module in both plant immunity and growth/development.
Southern corn leaf blight (SCLB), caused by Bipolaris maydis, is one of the most devastating diseases affecting maize production. However, only one SLCB resistance gene, conferring partial resistance, is currently known, underscoring the importance of isolating new SCLB resistance-related genes. Here, we performed a comparative proteomic analysis and identified 258 proteins showing differential abundance during the maize response to B. maydis. These proteins included an ascorbate peroxidase (Zea mays ascorbate peroxidase 1 (ZmAPX1)) encoded by a gene located within the mapping interval of a previously identified quantitative trait locus associated with SCLB resistance. ZmAPX1 overexpression resulted in lower H2O2 accumulation and enhanced resistance against B. maydis. Jasmonic acid (JA) contents and transcript levels for JA biosynthesis and responsive genes increased in ZmAPX1-overexpressing plants infected with B. maydis, whereas Zmapx1 mutants showed the opposite effects. We further determined that low levels of H2O2 are accompanied by an accumulation of JA that enhances SCLB resistance. These results demonstrate that ZmAPX1 positively regulates SCLB resistance by decreasing H2O2 accumulation and activating the JA-mediated defense signaling pathway. This study identified ZmAPX1 as a potentially useful gene for increasing SCLB resistance. Furthermore, the generated data may be relevant for clarifying the functions of plant APXs.
Plant immunity must be tightly controlled to avoid activation of defense mechanisms in the absence of pathogen attack. Protein phosphorylation is a common mechanism regulating immune signaling. In Arabidopsis thaliana, nine members of the type one protein phosphatase (TOPP) family (also known as protein phosphatase 1, PP1) have been identified. Here, we characterized the autoimmune phenotype of topp4‐1, a previously identified dominant‐negative mutant of TOPP4. Epistasis analysis showed that defense activation in topp4‐1 depended on NON‐RACE‐SPECIFIC DISEASE RESISTANCE1, PHYTOALEXIN DEFICIENT4, and the salicylic acid pathway. We generated topp1/4/5/6/7/8/9 septuple mutants to investigate the function of TOPPs in plant immunity. Elevated defense gene expression and enhanced resistance to Pseudomonas syringae pv. tomato (Pst) DC3000 in the septuple mutant indicate that TOPPs function in plant defense responses. Furthermore, TOPPs physically interacted with mitogen‐activated protein kinases (MAPKs) and affected the MAPK‐mediated downstream defense pathway. Thus, our study reveals that TOPPs are important regulators of plant immunity.
Pathogen avirulence (Avr) effectors interplay with corresponding plant resistance (R) proteins and activate robust plant immune responses. Although the expression pattern of Avr genes has been tied to their functions for a long time, it is still not clear how Avr gene expression patterns impact plant‐microbe interactions. Here, we selected PsAvr3b, which shows a typical effector gene expression pattern from a soybean root pathogen Phytophthora sojae. To modulate gene expression, we engineered PsAvr3b promoter sequences by in situ substitution with promoter sequences from Actin (constitutive expression), PsXEG1 (early expression), and PsNLP1 (later expression) using the CRISPR/Cas9. PsAvr3b driven by different promoters resulted in distinct expression levels across all the tested infection time points. Importantly, those mutants with low PsAvr3b expression successfully colonized soybean plants carrying the cognate R gene Rps3b. To dissect the difference in plant responses to the PsAvr3b expression level, we conducted RNA‐sequencing of different infection samples at 24 h postinfection and found soybean immune genes, including a few previously unknown genes that are associated with resistance. Our study highlights that fine‐tuning in Avr gene expression impacts the compatibility of plant disease and provides clues to improve crop resistance in disease control management.
The brown planthopper (BPH) and striped stem borer (SSB) are the most devastating insect pests in rice (Oryza sativa ) producing areas. Screening for endogenous resistant genes is the most practical strategy for rice insect‐resistance breeding. Forty‐five mutants showing high resistance against BPH were identified in a rice T‐DNA insertion population (11,000 putative homozygous lines) after 4 years of large‐scale field BPH‐resistance phenotype screening. Detailed analysis showed that deficiency of rice mitochondrial outer membrane protein 64 (OM64 ) gene resulted in increased resistance to BPH. Mitochondrial outer membrane protein 64 protein is located in the outer mitochondrial membrane by subcellular localization and its deficiency constitutively activated hydrogen peroxide (H2O2) signaling, which stimulated antibiosis and tolerance to BPH. The om64 mutant also showed enhanced resistance to SSB, a chewing insect, which was due to promotion of Jasmonic acid biosynthesis and related responses. Importantly, om64 plants presented no significant changes in rice yield‐related characters. This study confirmed OM64 as a negative regulator of rice herbivore resistance through regulating H2O2 production. Mitochondrial outer membrane protein 64 is a potentially efficient candidate to improve BPH and SSB resistance through gene deletion. Why the om64 mutant was resistant to both piercing‐sucking and chewing insects via a gene deficiency in mitochondria is discussed.
Rice OsLIC encoding a CCCH zinc finger transcription factor plays an important role in immunity. However, the immune signaling pathways that OsLIC-involved and the underlying mechanisms that OsLIC-conferred resistance against pathogens are largely unclear. Here, we show that OsLIC, as a substrate for OsMAPK6, negatively regulates resistance to Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc) by directly suppressing OsWRKY30 transcription. Biochemical assays showed that OsLIC bound to OsWRKY30 promoter and suppressed its transcription. Genetic assays confirmed that the osilc knockout mutants and OsWRKY30-overexpressing plants exhibited enhanced resistance to Xoo and Xoc, knocking out OsWRKY30 in the oslic mutants attenuated the resistance against bacterial pathogens. OsMAPK6 physically interacted with and phosphorylated OsLIC leading to decreased OsLIC DNA-binding activity, therefore, overexpression of OsLIC partially suppressed OsMAPK6-mediated rice resistance. In addition, both OsMAPK6-phosphorylated activation of OsLIC and phosphorylation-mimic OsLIC5D had reduced DNA-binding activity towards OsWRKY30 promoter, thereby promoting OsWRKY30 transcription. Collectively, these results reveal that OsMAPK6-mediated phosphorylation of OsLIC positively regulates rice resistance to Xoo and Xoc by modulating OsWRKY30 transcription, suggesting that OsMAPK6-OsLIC-OsWRKY30 module is an immune signaling pathway in response to the bacterial pathogens.
One of the most promising tools for the control of fungal plant diseases is spray-induced gene silencing (SIGS). In SIGS, small interfering RNA (siRNA) or double-stranded RNA (dsRNA) targeting essential or virulence-related pathogen genes are exogenously applied to plants and postharvest products to trigger RNA interference (RNAi) of the targeted genes, inhibiting fungal growth and disease. However, SIGS is limited by the unstable nature of RNA under environmental conditions. The use of layered double hydroxide or clay particles as carriers to deliver biologically active dsRNA, a formulation termed BioClay™, can enhance RNA durability on plants, prolonging its activity against pathogens. Here, we demonstrate that dsRNA delivered as BioClay can prolong protection against Botrytis cinerea, a major plant fungal pathogen, on tomato leaves and fruit and on mature chickpea plants. BioClay increased the protection window from 1 to 3 weeks on tomato leaves and from 5 to 10 days on tomato fruits, when compared with naked dsRNA. In flowering chickpea plants, BioClay provided prolonged protection for up to 4 weeks, covering the critical period of poding, whereas naked dsRNA provided limited protection. This research represents a major step forward for the adoption of SIGS as an eco-friendly alternative to traditional fungicides.
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