A critical role of a plant-specific TFIIB-related protein, BRP1, in salicylic acid-mediated immune response.

An integral part of plant immunity is transcription reprogramming by concerted action of specific transcription factors that activate or repress genes through recruitment or release of RNA polymerase II (Pol II). Pol II is assembled into Pol II holoenzyme at the promoters through association with a group of general transcription factors including transcription factor IIB (TFIIB) to activate transcription. Unlike other eukaryotic organisms, plants have a large family of TFIIB-related proteins with 15 members in Arabidopsis including several plant-specific TFIIB-related proteins (BRPs). Molecular genetic analysis has revealed important roles of some BRPs in plant reproductive processes. In this study, we report that Arabidopsis knockout mutants for BRP1, the founding member of the BRP protein family, were normal in growth and development, but were hypersusceptible to the bacterial pathogen Psuedomonas syringae. The enhanced susceptibility of the brp1 mutants was associated with reduced expression of salicylic acid (SA) biosynthetic gene ISOCHORISMATE SYNTHASE 1 (ICS1) and SA-responsive PATHOGENESIS-RELATED (PR) genes. Pathogen-induced SA accumulation was reduced in the brp1 mutants and exogenous SA rescued the brp1 mutants for resistance to the bacterial pathogen. In uninfected plants, BRP1 was primarily associated with the plastids but pathogen infection induced its accumulation in the nucleus. BRP1 acted as a transcription activator in plant cells and binded to the promoter of ICS1. These results collectively indicate that BRP1 is a functionally specialized transcription factor that increasingly accumulates in the nucleus in response to pathogen infection to promote defense gene expression.

Enhancement of broad-spectrum disease resistance in wheat through key genes involved in systemic acquired resistance.

Systemic acquired resistance (SAR) is an inducible disease resistance phenomenon in plant species, providing plants with broad-spectrum resistance to secondary pathogen infections beyond the initial infection site. In Arabidopsis, SAR can be triggered by direct pathogen infection or treatment with the phytohormone salicylic acid (SA), as well as its analogues 2,6-dichloroisonicotinic acid (INA) and benzothiadiazole (BTH). The SA receptor non-expressor of pathogenesis-related protein gene 1 (NPR1) protein serves as a key regulator in controlling SAR signaling transduction. Similarly, in common wheat (Triticum aestivum), pathogen infection or treatment with the SA analogue BTH can induce broad-spectrum resistance to powdery mildew, leaf rust, Fusarium head blight, and other diseases. However, unlike SAR in the model plant Arabidopsis or rice, SAR-like responses in wheat exhibit unique features and regulatory pathways. The acquired resistance (AR) induced by the model pathogen Pseudomonas syringae pv. tomato strain DC3000 is regulated by NPR1, but its effects are limited to the adjacent region of the same leaf and not systemic. On the other hand, the systemic immunity (SI) triggered by Xanthomonas translucens pv. cerealis (Xtc) or Pseudomonas syringae pv. japonica (Psj) is not controlled by NPR1 or SA, but rather closely associated with jasmonate (JA), abscisic acid (ABA), and several transcription factors. Furthermore, the BTH-induced resistance (BIR) partially depends on NPR1 activation, leading to a broader and stronger plant defense response. This paper provides a systematic review of the research progress on SAR in wheat, emphasizes the key regulatory role of NPR1 in wheat SAR, and summarizes the potential of pathogenesis-related protein (PR) genes in genetically modifying wheat to enhance broad-spectrum disease resistance. This review lays an important foundation for further analyzing the molecular mechanism of SAR and genetically improving broad-spectrum disease resistance in wheat.

Complete genome sequencing and construction of full-length infectious cDNA clone of papaya ringspot virus-HYD isolate and its efficient in planta expression.

Papaya ringspot virus (PRSV) is a devastating Potyvirus that causes papaya ringspot disease in Carica papaya plantations globally. In this study, the complete genome sequence of a PRSV isolate from Shankarpalli, Telangana, India, was reported and designated as PRSV-HYD (KP743981.1). The genome is a single-stranded positive-sense RNA comprising 10,341 nucleotides. Phylogenetic analysis revealed that PRSV-HYD is closely related to PRSV Pune (Aundh) isolate with 92 and 95% nucleotide and amino acid sequence identity, respectively. To develop infectious cDNA (icDNA), the complete nucleotide sequence of PRSV-HYD was cloned between the right and left borders in the binary vector pCB301 using BglII and XmaI restriction sites. Cauliflower mosaic virus (CaMV) double promoter (35S) was fused at the 5'-end and Avocado sunblotch viroid (ASBVd) ribozyme (RZ) sequence was fused to the 3' end to generate an authentic 3' viral end in the transcribed mRNAs. The icDNA generated was mobilized into the Agrobacterium tumefaciens EHA 105, and the agrobacterial cultures were infiltrated into the natural host C. papaya and a non-host Nicotiana benthamiana plants; both did not show any symptoms. In RT-PCR analysis of RNAs isolated from N. benthamiana, we could detect viral genes as early as 3 days and continued up to 28 days post infiltration. Alternatively, virion particles were purified from agroinfiltrated N. benthamiana plants and introduced into C. papaya by mechanical inoculation as well as by pinprick method. In both cases, we could see visible systemic symptoms similar to that of wild type by 40 days. Additionally, we studied the expression patterns of the genes related to plant defense, transcription factors (TFs), and developmental aspects from both C. papaya and N. benthamiana.

Transcriptomic and Functional Analyses Reveal the Different Roles of Vitamins C, E, and K in Regulating Viral Infections in Maize.

Maize lethal necrosis (MLN), one of the most important maize viral diseases, is caused by maize chlorotic mottle virus (MCMV) infection in combination with a potyvirid, such as sugarcane mosaic virus (SCMV). However, the resistance mechanism of maize to MLN remains largely unknown. In this study, we obtained isoform expression profiles of maize after SCMV and MCMV single and synergistic infection (S + M) via comparative analysis of SMRT- and Illumina-based RNA sequencing. A total of 15,508, 7567, and 2378 differentially expressed isoforms (DEIs) were identified in S + M, MCMV, and SCMV libraries, which were primarily involved in photosynthesis, reactive oxygen species (ROS) scavenging, and some pathways related to disease resistance. The results of virus-induced gene silencing (VIGS) assays revealed that silencing of a vitamin C biosynthesis-related gene, ZmGalDH or ZmAPX1, promoted viral infections, while silencing ZmTAT or ZmNQO1, the gene involved in vitamin E or K biosynthesis, inhibited MCMV and S + M infections, likely by regulating the expressions of pathogenesis-related (PR) genes. Moreover, the relationship between viral infections and expression of the above four genes in ten maize inbred lines was determined. We further demonstrated that the exogenous application of vitamin C could effectively suppress viral infections, while vitamins E and K promoted MCMV infection. These findings provide novel insights into the gene regulatory networks of maize in response to MLN, and the roles of vitamins C, E, and K in conditioning viral infections in maize.

Maize Phytocytokines Modulate Pro-Survival Host Responses and Pathogen Resistance.

Phytocytokines are signaling peptides that alert plant cells of danger. However, the downstream responses triggered by phytocytokines and their effect on plant survival are still largely unknown. Here, we have identified three biologically active maize orthologues of phytocytokines previously described in other plants. The maize phytocytokines show common features with microbe-associated molecular patterns (MAMPs), including the induction of immune-related genes and activation of papain-like cysteine proteases. In contrast to MAMPs, phytocytokines do not promote cell death in the presence of wounding. In infection assays with two fungal pathogens, we found that phytocytokines affect the development of disease symptoms, likely due to the activation of phytohormonal pathways. Collectively, our results show that phytocytokines and MAMPs trigger unique and antagonistic features of immunity. We propose a model in which phytocytokines activate immune responses partially similar to MAMPs but, in contrast to microbial signals, they act as danger and survival molecules to the surrounding cells. Future studies will focus on the components determining the divergence of signaling outputs upon phytocytokine activation. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Additive Effect of the Composition of Endophytic Bacteria Bacillus subtilis on Systemic Resistance of Wheat against Greenbug Aphid Schizaphis graminum Due to Lipopeptides.

The use of biocontrol agents based on endophytic bacteria against phloem-feeding insects is limited by a lack of knowledge and understanding of the mechanism of action of the endophyte community that makes up the plant microbiome. In this work, the mechanisms of the additive action of endophytic strains B. subtilis 26D and B. subtilis 11VM on the resistance of bread spring wheat against greenbug aphid Schizaphis graminum, was studied. It was shown that B. subtilis 26D secreted lipopeptide surfactin and phytohormones cytokinins, and B. subtilis 11VM produced iturin and auxins into the cultivation medium. Both strains and their lipopeptide-rich fractions showed direct aphicidal activity against greenbug aphid. For the first time, it was shown that B. subtilis 26D and B. subtilis 11VM in the same manner, as well as their lipopeptide-rich fractions, activated the expression of salicylate- and ethylene-dependent PR genes, and influenced plant redox metabolism, which led to an increase in plant endurance against aphids. The composition of endophytic strains B. subtilis 26D + B. subtilis 11VM had an additive effect on plant resistance to aphids due to an increase in the number of endophytic bacterial cells, and, as well as due to the synergistic effect of their mixture of lipopeptides - surfactin + iturin, both on the aphid mortality and on the expression of PR1 and PR3 genes. All these factors can be the reason for the observed increase in the growth of plants affected by aphids under the influence of B. subtilis 26D and B. subtilis 11VM, individually and in composition. The study demonstrates the possibility of creating in the future an artificial composition to enhance plant microbiome with endophytic bacteria, which combines growth-promoting and plant immunity stimulating properties against phloem-feeding insects. This direction is one of the most promising approaches to green pesticide discovery in the future.

Chitosan-Salicylic acid and Zinc sulphate nano-formulations defend against yellow rust in wheat by activating pathogenesis-related genes and enzymes.

Stripe rust instigated by Puccinia striiformis f. sp. tritici causes major yield loss in wheat. In this study, disease resistance was induced in wheat by pre-activation of pathogenesis related (PR) genes using two different nano-formulations (NFs) i.e. Chitosan- Salicylic acid (SA) NFs (CH-NFs) and Zinc sulphate NFs (Zn-NFs). These NFs were synthesized using green approach and were characterized using various techniques. Both NFs effectively controlled stripe rust in wheat genotypes (WH 711 and WH 1123) by significantly increasing activities of phenylalanine ammonia lyase, tyrosine ammonia lyase and polyphenol oxidase enzymes when compared with disease free-control and diseased plants. Total soluble sugar (TSS) level was highest in CH-NF treated plants. TSS was also relatively higher in diseased plants than disease free-control as well as Zn-NF treated plants. Both CH-NFs and Zn-NFs induced the expression of PR genes. In CH-NF treated plants, the relative expression of PR genes was higher on the 3rd day after spraying (DAS) of NFs as compared to diseased and Zn-NF treated plants in both the genotypes. While in case of Zn-NF treated plants, relative expression of PR genes was higher on 5th DAS as compared to diseased and disease free-control plants. Early rise in expression of PR genes due to NF treatments was responsible for disease resistance in both the wheat genotypes as evidenced by a lower average coefficient of infection. These NFs can be synthesized easily with low cost input, are eco-friendly and can be effectively used against yellow rust as well as other wheat diseases.

Lactic Acid Bacteria as Potential Biocontrol Agents for Fusarium Head Blight Disease of Spring Barley.

Fusarium head blight (FHB) is a devastating disease encountered by spring-grown barley. Traditionally, synthetic chemicals have been used to control this disease on small grain cereals. A move toward biological control agents as part of sustainable agriculture is pertinent due to the evolutionary mechanisms employed by fungal diseases to circumvent current protection strategies. This study evaluated the effect of six lactic acid bacteria isolates on the development of FHB under in vitro and glasshouse conditions. The relative expression of Fusarium marker genes and transcription factors under Fusarium infection was examined. Dual-culture assays observed inhibition zones of up to 10 and 17% of total plate area for L. amylovorus FST 2.11 and L. brevis R2Δ, respectively. Detached leaf assays validated the antifungal activity and showed the potential of all test isolates to significantly inhibit sporulation of Fusarium culmorum and Fusarium graminearum strains. Spray inoculation of lactic acid bacteria to barley spikelets prior to Fusarium spore application significantly reduced disease severity for five candidates (P < 0.05) under glasshouse conditions. Mycotoxin analysis revealed the ability of L. amylovorus DSM20552 to significantly reduce deoxynivalenol content in spikelets (P < 0.05). A preliminary gene expression study showed the positive influence of lactic acid bacteria on the expression of important defense-related marker genes and transcription factors upon FHB. These results indicate the potential of lactic acid bacteria to be included as part of an integrated pest management strategy for the management of FHB disease. This strategy will reduce FHB severity and deoxynivalenol (DON) contamination of spring barley, leading to high acceptance in the grain market.

Novel insights into host specificity of Pyricularia oryzae and Pyricularia grisea in the infection of gramineous plant roots.

Pyricularia oryzae and Pyricularia grisea are pathogens that cause blast disease in various monocots. It has been reported that P. oryzae infects the leaves and roots of rice via different mechanisms. However, it is unclear to what extent the tissue types affect the host specificities of P. oryzae and P. grisea. Here, we evaluated the tissue-specific infection strategies of P. oryzae and P. grisea in various gramineous plants. Generally, mycelial plug inoculation caused root browning but the degree of browning did not simply follow the disease index on leaves. Interestingly, the Triticum and Digitaria pathotypes caused strong root growth inhibition in rice, wheat, and barley. Moreover, the Digitaria pathotype inhibited root branching only in rice. Culture filtrate reproduced these inhibitory effects on root, suggesting that some secreted molecules are responsible for the inhibitions. Observation of root sections revealed that most of the infection hyphae penetrated intercellular spaces and further extended into root cells, regardless of pathotype and host plant. The infection hyphae of Digitaria and Triticum pathotypes tended to localize in the outer layer of rice roots, but not in those of wheat and barley roots. The infection hyphae of the Oryza pathotype were distributed in both the intercellular and intracellular spaces of rice root cells. Pathogenesis-related genes and reactive oxygen species accumulation were induced after root inoculation with all combinations. These results suggest that resistance reactions were induced in the roots of gramineous plants against the infection with Pyricularia isolates but failed to prevent fungal invasion.

Induce defense response of DADS in eggplants during the biotrophic phase of Verticillium dahliae.

PURPOSE: Verticillium wilt is a destructive vascular disease in eggplants. The complex defensive mechanisms of eggplant against this disease are very limited. METHODS: Our work examined the bioactive properties of garlic allelochemical diallyl disulfide (DADS) as potential biostimulants for defense against V. dahliae in eggplant seedlings. We, therefore, foliar sprayed DADS on eggplants to study the defense response during the early biotrophic phase of V. dahliae (a hemibiotroph). RESULTS: DADS application significantly increased root peroxidase (POD), phenylalanine-ammonia lyase (PAL) enzyme activity, and reduced H2O2 levels after 24 h of fungal inoculation. Salicylic acid (SA) in leaves and roots was significantly increased while, the jasmonic acid (JA), indole acetic acid (IAA), and abscisic acid (ABA) levels were decreased. The microscopic examinations of V. dahliae infection in roots displayed that the progression of infection was restricted in DADS-treated plants. Depositions of lignin and phenolic compounds such as ferulic acid, p-coumaric acid, and caffeic acid content were significantly higher in DADS-treated plants at 48 h post-inoculation. Similarly, the DADS application up-regulated pathogenesis-related (PR1, PR2, and PR5), mitogen-activated protein kinase (MPK1), and lipoxygenase (LOX) genes. Furthermore, DADS-treated plants exhibited a lower disease severity index (23.3% vs. 57.0% in controls), indicating successful defense against V. dahliae. CONCLUSIONS: Our findings concluded that the biological function of garlic allelochemical DADS has a prominent role in the higher defense resistance of eggplants during the early infection of V. dahliae.

Identification of novel resources for panicle blast resistance from wild rice accessions and mutants of cv. Nagina 22 by syringe inoculation under field conditions.

Panicle blast is the most severe type of rice blast disease. Screening of rice genotypes for panicle blast resistance at the field level requires an efficient and robust method of inoculation. Here, we standardized a method that can be utilized for both small- and large-scale screening and assessment of panicle blast infection and disease reaction. The method involves inoculation of Magnaporthe oryzae spore culture in the neck of the rice panicle using a syringe and covering the inoculation site with wet cotton wrapped with aluminum foil to provide the required humidity for spore germination. The method was standardized using panicle blast-resistant cv. Tetep and susceptible cv. HP2216 inoculated with Mo-ni-025 isolate of M. oryzae. The method was evaluated at phenotypic as well as molecular level by expression analysis of disease responsive pathogenesis-related (PR) genes. We found this method simple, robust, reliable, and highly efficient for screening of large germplasm sets of rice for panicle blast. This was validated by screening the wild rice germplasm for panicle blast response in the field using three M. oryzae strains and subsequently with the most virulent strain in 45 EMS-induced mutants of Nagina 22 shortlisted based on field screening in a blast hotspot region. We identified five novel blast disease-resistant wild rice genotypes and 15 Nagina 22 mutants that can be used in breeding programmes.

Trichoderma-Induced Resistance to Botrytis cinerea in Solanum Species: A Meta-Analysis.

With the idea of summarizing the outcomes of studies focusing on the resistance induced by Trichoderma spp. against Botrytis cinerea in tomato, the present paper shows, for the first time, results of a meta-analysis performed on studies published from 2010 to 2021 concerning the cross-talk occurring in the tomato-Trichoderma-B. cinerea system. Starting from an initial set of 40 papers, the analysis was performed on 15 works and included nine parameters, as a result of a stringent selection mainly based on the availability of more than one article including the same indicator. The resulting work not only emphasizes the beneficial effects of Trichoderma in the control of grey mold in tomato leaves (reduction in disease intensity, severity and incidence and modulation of resistance genes in the host), but carefully drives the readers to reply to two questions: (i) What are the overall effects of Trichoderma on B. cinerea infection in tomato? (ii) Do the main effects of Trichoderma differ based on the tomato species, Trichoderma species, amount, type and duration of treatment? At the same time, this meta-analysis highlights some weak points of the available literature and should be seen as an invitation to improve future works to better the conceptualization and measure.

The small GTPase NtRHO1 negatively regulates tobacco defense response to tobacco mosaic virus by interacting with NtWRKY50.

Small GTPases play critical roles in the regulation of plant growth and development. However, the mechanism of action of small GTPases in plant response to virus infection remains largely unknown. Here, the gene encoding a Rho-type GTPase, NtRHO1, was identified as one of the genes up-regulated after tobacco mosaic virus (TMV) infection. Subcellular localization of NtRHO1 showed that it was located in the cytoplasm, plasma membrane, and nucleus. Transient overexpression of NtRHO1 in Nicotiana benthamiana accelerated TMV reproduction and led to the production of reactive oxygen species. By contrast, silencing of NtRHO1 reduced the sensitivity of N. benthamiana to TMV-GFP. Further exploration revealed a direct interaction between NtRHO1 and NtWRKY50, a positive regulator of the N. benthamiana response to virus infection. Yeast one-hybrid and electrophoretic mobility shift assays showed that this regulation was related to the capacity of NtWRKY50 to bind to the WK-box of the PR1 promoter, which was weakened by the interaction between NtRHO1 and NtWRKY50. Thus, our results indicate that the small GTPase NtRHO1 plays a negative role in tobacco response to TMV infection by interacting with transcription factor NtWRKY50, resulting in reduced plant immunity.

Evolution of the Sex Pheromone Communication System in Ostrinia Moths.

It remains a conundrum in the evolution of sexual communication how the signals and responses can co-ordinate the changes during speciation. The genus Ostrinia contains several closely related species as well as distinctive strains with pheromone polymorphism and represents an example of ongoing speciation. Extensive studies in the genus, especially in the species the European corn borer O. nubilalis (ECB), the Asian corn borer O. furnacalis (ACB) and the adzuki bean borer O. scapulalis (ABB), have provided valuable insights into the evolution of sex pheromone communication. This review presents a comprehensive overview of the research on pheromone communication in different Ostrinia species over the past four decades, including pheromone identification and biosynthesis, the ligand profiles of pheromone receptor (PR) genes, the physiology of peripheral olfactory sensory neurons (OSNs) and the projection pattern to the antennal lobe. By integrating and comparing the closely related Ostrinia species and strains, it provides an evolutionary perspective on the sex pheromone communication in moths in general and also outlines the outstanding questions that await to be elucidated by future studies.

Silicon supplementation improves early blight resistance in Lycopersicon esculentum Mill. by modulating the expression of defense-related genes and antioxidant enzymes.

Early blight is the most devastating disease in tomato which causes huge yield losses across the globe. Hence, development of specific, efficient and ecofriendly tools are required to increase the disease resistance in tomato plants. Here, we systematically investigate the defensive role and priming effect of silicon (Si) in tomato plants under control and infected conditions. Based on the results, Si-treated tomato plants showed improved resistance to Alternaria solani as there was delay in symptoms and reduced disease severity than non-Si-treated plants. To further examine the Si-mediated molecular priming in tomato plants, expression profiling of defense-related genes like PR1, PR2, WRKYII, PR3, LOXD and JERF3 was studied in control, Si-supplemented, A. solani-inoculated and Si + A. solani-inoculated plants. Interestingly, Si significantly increased the expression of jasmonic acid (JA) marker genes (PR3, LOXD and JERF3) than salicylic acid (SA) marker genes (PR1, PR2 and WRKYII). However, Si + A. solani-inoculated plants showed higher expression levels of defence genes except WRKYII than A. solani-inoculated or Si-treated plants. Furthermore, pre-supplementation of Si to A. solani-infected tomato plants showed increased activity of antioxidant enzymes viz. superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) and peroxidase (POD) than control, Si-treated and A. solani-inoculated plants. Altogether, present study highlights the defensive role of Si in tomato plants in response to A. solani by increasing not only the transcript levels of defense signature genes, but also the activity of antioxidant enzymes.

The Medicago truncatula Sugar Transport Protein 13 and Its Lr67res-Like Variant Confer Powdery Mildew Resistance in Legumes via Defense Modulation.

Obligate biotrophic pathogens like the pea powdery mildew© (PM) Erysiphe pisi establish long-term feeding relationships with their host, during which they siphon sugars from host cells through haustoria. Plants in turn deploy sugar transporters to restrict carbon allocation toward pathogens, as a defense mechanism. Studies in Arabidopsis have shown that sugar transport protein 13 (STP13), a proton-hexose symporter involved in apoplasmic hexose retrieval, contributes to bacterial and necrotrophic fungal resistance by limiting sugar flux toward these pathogens. By contrast, expression of Lr67res,a transport-deficient wheat STP13 variant harboring two amino acid substitutions (G144R and V387L), conferred resistance against biotrophic fungi in wheat and barley, indicating its broad applicability in disease management. Here, we investigated the role of STP13 and STP13G144R in legume-PM interactions. We show that Medicago truncatula STP13.1 is a proton-hexose symporter involved in basal resistance against PM and indirectly show that Lr67res-mediated PM resistance, so far reported only in monocots, is transferable to legumes. Among the 30 MtSTPs, STP13.1 exhibited the highest fold induction in PM-challenged leaves and was also responsive to chitosan, ABA and sugar treatment. Functional assays in yeast showed that introduction of the G144R mutation but not V388L abolished MtSTP13.1's hexose uptake ability. Virus-induced gene silencing of MtSTP13 repressed pathogenesis-related (PR) gene expression and enhanced PM susceptibility in M. truncatula whereas transient overexpression of MtSTP13.1 or MtSTP13.1G144R in pea induced PR and isoflavonoid pathway genes and enhanced PM resistance. We propose a model in which STP13.1-mediated sugar signaling triggers defense responses against PM in legumes.

Differential regulation of the durum wheat Pathogenesis-related protein (PR1) by Calmodulin TdCaM1.3 protein.

In plants, pathogenesis-related 1 protein (PR1) is considered as important defense protein. The production and accumulation of PR proteins in plants are one of the important responses to several biotic and abiotic stresses. In this regard, PR1 gene was isolated from Triticum turgidum ssp durum and was named as TdPR1.2. The amino acid sequence of TdPR1.2 protein showed 100%, 97.13%, and 44.41% with known PR1 proteins isolated from Triticum aestivum TdPR1-18, PRB1.2 of Aegilops tauschii subsp. tauschii and Arabidopsis thaliana respectively. qRT-PCR showed that TdPR1.2 was induced specially in leaves of durum wheat treated with Salicylic acid for 48 h. Besides, bioinformatic analysis showed that the durum wheat TdPR1.2 harbors a calmodulin binding domain located in it's C-terminal part and that this domain is conserved among different PR1 proteins isolated so far. However, no information is available about the regulation of PR genes by calmodulin and Ca2+ complex (CaM/Ca2+). Here, we showed that TdPR1.2 gene exhibits an antibacterial effect as revealed by the in vitro tests against 8 different bacteria and against the fungi Septoria tritici. In addition, we demonstrate for the first time that PR1 proteins are able to bind to CaM in a Ca2+-dependent manner via a GST-Pull down assay. Finally, in presence of Mn2+ cations, CaM/Ca2+ complex stimulated the antimicrobial effect of TdPR1.2. Such effects were not reported so far, and raise a possible role for CaM/Ca2+ complex in the regulation of plant PRs during cellular response to external signals.

Priming by Timing: Arabidopsis thaliana Adjusts Its Priming Response to Lepidoptera Eggs to the Time of Larval Hatching.

Plants can respond to eggs laid by herbivorous insects on their leaves by preparing (priming) their defense against the hatching larvae. Egg-mediated priming of defense is known for several plant species, including Brassicaceae. However, it is unknown yet for how long the eggs need to remain on a plant until a primed defense state is reached, which is ecologically manifested by reduced performance of the hatching larvae. To address this question, we used Arabidopsis thaliana, which carried eggs of the butterfly Pieris brassicae for 1-6 days prior to exposure to larval feeding. Our results show that larvae gained less biomass the longer the eggs had previously been on the plant. The strongest priming effect was obtained when eggs had been on the plant for 5 or 6 days, i.e., for (almost) the entire development time of the Pieris embryo inside the egg until larval hatching. Transcript levels of priming-responsive genes, levels of jasmonic acid-isoleucine (JA-Ile), and of the egg-inducible phytoalexin camalexin increased with the egg exposure time. Larval performance studies on mutant plants revealed that camalexin is dispensable for anti-herbivore defense against P. brassicae larvae, whereas JA-Ile - in concert with egg-induced salicylic acid (SA) - seems to be important for signaling egg-mediated primed defense. Thus, A. thaliana adjusts the kinetics of its egg-primed response to the time point of larval hatching. Hence, the plant is optimally prepared just in time prior to larval hatching.

Differential Quantitative Requirements for NPR1 Between Basal Immunity and Systemic Acquired Resistance in Arabidopsis thaliana.

Non-expressor of pathogenesis-related (PR) genes1 (NPR1) is a key transcription coactivator of plant basal immunity and systemic acquired resistance (SAR). Two mutant alleles, npr1-1 and npr1-3, have been extensively used for dissecting the role of NPR1 in various signaling pathways. However, it is unknown whether npr1-1 and npr1-3 are null mutants. Moreover, the NPR1 transcript levels are induced two- to threefold upon pathogen infection or salicylic acid (SA) treatment, but the biological relevance of the induction is unclear. Here, we used molecular and biochemical approaches including quantitative PCR, immunoblot analysis, site-directed mutagenesis, and CRISPR/Cas9-mediated gene editing to address these questions. We show that npr1-3 is a potential null mutant, whereas npr1-1 is not. We also demonstrated that a truncated npr1 protein longer than the hypothesized npr1-3 protein is not active in SA signaling. Furthermore, we revealed that TGACG-binding (TGA) factors are required for NPR1 induction, but the reverse TGA box in the 5'UTR of NPR1 is dispensable for the induction. Finally, we show that full induction of NPR1 is required for basal immunity, but not for SAR, whereas sufficient basal transcription is essential for full-scale establishment of SAR. Our results indicate that induced transcript accumulation may be differentially required for different functions of a specific gene. Moreover, as npr1-1 is not a null mutant, we recommend that future research should use npr1-3 and potential null T-DNA insertion mutants for dissecting NPR1's function in various physiopathological processes.

ATG4 Mediated Psm ES4326/AvrRpt2-Induced Autophagy Dependent on Salicylic Acid in Arabidopsis Thaliana.

Psm ES4326/AvrRpt2 (AvrRpt2) was widely used as the reaction system of hypersensitive response (HR) in Arabidopsis. The study showed that in npr1 (GFP-ATG8a), AvrRpt2 was more effective at inducing the production of autophagosome and autophagy flux than that in GFP-ATG8a. The mRNA expression of ATG1, ATG6 and ATG8a were more in npr1 during the early HR. Based on transcriptome data analysis, enhanced disease susceptibility 1 (EDS1) was up-regulated in wild-type (WT) but was not induced in atg4a4b (ATG4 deletion mutant) during AvrRpt2 infection. Compared with WT, atg4a4b had higher expression of salicylic acid glucosyltransferase 1 (SGT1) and isochorismate synthase 1 (ICS1); but less salicylic acid (SA) in normal condition and the same level of free SA during AvrRpt2 infection. These results suggested that the consumption of free SA should be occurred in atg4a4b. AvrRpt2 may trigger the activation of Toll/Interleukin-1 receptor (TIR)-nucleotide binding site (NB)-leucine rich repeat (LRR)-TIR-NB-LRR-to induce autophagy via EDS1, which was inhibited by nonexpressor of PR genes 1 (NPR1). Moreover, high expression of NPR3 in atg4a4b may accelerate the degradation of NPR1 during AvrRpt2 infection.