Potato miR394 targets StA/N-INVE and StLCR to negatively regulate late blight resistance.

MicroRNAs (miRNAs) are small noncoding RNAs in eukaryotes. Plant endogenous miRNAs play pivotal roles in regulating plant development and defense responses. MicroRNA394 (miR394) has been reported to regulate plant development, abiotic stresses and defense responses. Previous reports showed that miR394 responded to P. infestans inoculation in potato, indicating that miR394 may be involved in defense responses. In this study, we further investigated its role in potato defense against P. infestans. Stable expression of miR394 in tobacco and potato enhances the susceptibility to P. infestans, which is accompanied with the reduced accumulation of ROS and down-regulation of the PTI (pattern-triggered immunity) marker genes. Besides well-known target StLCR, miR394 also targets StA/N-INVE, which encodes a chloroplast Alkaline/Neutral Invertases (A/N-INVE). Both StLCR and StA/N-INVE positively regulate late blight resistance, while miR394 degrades them. Interestingly, StA/N-INVE is located in the chloroplast, indicating that miR394 may manipulate chloroplast immunity. Degradation of StA/N-INVE may affect the chloroplast function and hence lead to the compromised ROS (reactive oxygen species) burst and reduced retrograde signaling from the chloroplast to the nucleus and cytoplasm. In summary, this study provides new information that miR394 targets and degrades StA/N-INVE and StLCR, which are positive regulators, to enhance potato susceptibility to P. infestans.

Dynamic crosstalk between silicon nanomaterials and potentially toxic trace elements in plant-soil systems.

Agricultural soil pollution with potentially toxic trace elements (PTEs) has emerged as a significant environmental concern, jeopardizing food safety and human health. Although, conventional remediation approaches have been used for PTEs-contaminated soils treatment; however, these techniques are toxic, expensive, harmful to human health, and can lead to environmental contamination. Nano-enabled agriculture has gained significant attention as a sustainable approach to improve crop production and food security. Silicon nanomaterials (SiNMs) have emerged as a promising alternative for PTEs-contaminated soils remediation. SiNMs have unique characteristics, such as higher chemical reactivity, higher stability, greater surface area to volume ratio and smaller size that make them effective in removing PTEs from the environment. The review discusses the recent advancements and developments in SiNMs for the sustainable remediation of PTEs in agricultural soils. The article covers various synthesis methods, characterization techniques, and the potential mechanisms of SiNMs to alleviate PTEs toxicity in plant-soil systems. Additionally, we highlight the potential benefits and limitations of SiNMs and discusses future directions for research and development. Overall, the use of SiNMs for PTEs remediation offers a sustainable platform for the protection of agricultural soils and the environment.

LsNRL4 enhances photosynthesis and decreases leaf angles in lettuce.

Lettuce (Lactuca sativa) is one of the most important vegetables worldwide and an ideal plant for producing protein drugs. Both well-functioning chloroplasts that perform robust photosynthesis and small leaf angles that enable dense planting are essential for high yields. In this study, we used an F2 population derived from a cross between a lettuce cultivar with pale-green leaves and large leaf angles to a cultivar with dark-green leaves and small leaf angles to clone LsNRL4, which encodes an NPH3/RPT2-Like (NRL) protein. Unlike other NRL proteins in lettuce, the LsNRL4 lacks the BTB domain. Knockout mutants engineered using CRISPR/Cas9 and transgenic lines overexpressing LsNRL4 verified that LsNRL4 contributes to chloroplast development, photosynthesis and leaf angle. The LsNRL4 gene was not present in the parent with pale-green leaves and enlarged leaf angles. Loss of LsNRL4 results in the enlargement of chloroplasts, decreases in the amount of cellular space allocated to chloroplasts and defects in secondary cell wall biosynthesis in lamina joints. Overexpressing LsNRL4 significantly improved photosynthesis and decreased leaf angles. Indeed, the plant architecture of the overexpressing lines is ideal for dense planting. In summary, we identified a novel NRL gene that enhances photosynthesis and influences plant architecture. Our study provides new approaches for the breeding of lettuce that can be grown in dense planting in the open field or in modern plant factories. LsNRL4 homologues may also be used in other crops to increase photosynthesis and improve plant architecture.

A Phytophthora effector promotes homodimerization of host transcription factor StKNOX3 to enhance susceptibility.

Oomycete pathogens secrete hundreds of cytoplasmic RxLR effectors to modulate host immunity by targeting diverse plant proteins. Revealing how effectors manipulate host proteins is pivotal to understanding infection processes and to developing new strategies to control plant disease. Here we show that the Phytophthora infestans RxLR effector Pi22798 interacts in the nucleus with a potato class II knotted-like homeobox (KNOX) transcription factor, StKNOX3. Silencing the ortholog NbKNOX3 in Nicotiana benthamiana reduces host colonization by P. infestans, whereas transient and stable overexpression of StKNOX3 enhances infection. StKNOX3 forms a homodimer which is dependent on its KNOX II domain. The KNOX II domain is also essential for Pi22798 interaction and for StKNOX3 to enhance P. infestans colonization, indicating that StKNOX3 homodimerization contributes to susceptibility. However, critically, the effector Pi22798 promotes StKNOX3 homodimerization, rather than heterodimerization to another KNOX transcription factor StKNOX7. These results demonstrate that the oomycete effector Pi22798 increases pathogenicity by promoting homodimerization specifically of StKNOX3 to enhance susceptibility.

Phytophthora infestans RXLR effector Pi04089 perturbs diverse defense-related genes to suppress host immunity.

BACKGROUND: The oomycete pathogen secretes many effectors into host cells to manipulate host defenses. For the majority of effectors, the mechanisms related to how they alter the expression of host genes and reprogram defenses are not well understood. In order to investigate the molecular mechanisms governing the influence that the Phytophthora infestans RXLR effector Pi04089 has on host immunity, a comparative transcriptome analysis was conducted on Pi04089 stable transgenic and wild-type potato plants. RESULTS: Potato plants stably expressing Pi04089 were more susceptible to P. infestans. RNA-seq analysis revealed that 658 upregulated genes and 722 downregulated genes were characterized in Pi04089 transgenic lines. A large number of genes involved in the biological process, including many defense-related genes and certain genes that respond to salicylic acid, were suppressed. Moreover, the comparative transcriptome analysis revealed that Pi04089 significantly inhibited the expression of many flg22 (a microbe-associated molecular pattern, PAMP)-inducible genes, including various Avr9/Cf-9 rapidly elicited (ACRE) genes. Four selected differentially expressed genes (StWAT1, StCEVI57, StKTI1, and StP450) were confirmed to be involved in host resistance against P. infestans when they were transiently expressed in Nicotiana benthamiana. CONCLUSION: The P. infestans effector Pi04089 was shown to suppress the expression of many resistance-related genes in potato plants. Moreover, Pi04089 was found to significantly suppress flg22-triggered defense signaling in potato plants. This research provides new insights into how an oomycete effector perturbs host immune responses at the transcriptome level.

Phytophthora infestans RXLR Effectors Target Parallel Steps in an Immune Signal Transduction Pathway.

The potato (Solanum tuberosum) blight pathogen Phytophthora infestans delivers Arg-X-Leu-Arg (RXLR) effector proteins into host cells to subvert plant immune responses and promote colonization. We show that transient expression and stable transgenic expression of the RXLR effector Pi22926 in Nicotiana benthamiana promotes leaf colonization by P. infestans. Pi22926 suppresses cell death triggered by coexpression of the Cladosporium fulvum avirulence protein Avr4 and the tomato (Solanum lycopersicum) resistance protein Cf4. Pi22926 interacts with a potato mitogen-activated protein kinase kinase kinase, StMAP3Kß2, in the nucleoplasm. Virus-induced gene silencing (VIGS) of the ortholog NbMAP3Kß2 in N. benthamiana enhances P. infestans colonization and attenuates Cf4/Avr4-induced cell death, indicating that this host protein is a positive regulator of immunity. Cell death induced by Cf4/Avr4 is dependent on NbMAP3Kε and NbMAP3Kß2, indicating that these MAP3Ks function in the same signaling pathway. VIGS of NbMAP3Kß2 does not compromise cell death triggered by overexpression of MAP3Kε. Similarly, VIGS of NbMAP3Kε does not attenuate cell death triggered by MAP3Kß2, demonstrating that these MAP3K proteins function in parallel. In agreement, Pi22926 or another RXLR effector, PexRD2, only suppresses cell death triggered by expression of StMAP3Kß2 or StMAP3Kε, respectively. Our data reveal that two P. infestans effectors, PexRD2 and Pi22926, promote P. infestans colonization by targeting MAP3K proteins that act in parallel in the same signal transduction pathway.

The oomycete microbe-associated molecular pattern Pep-13 triggers SERK3/BAK1-independent plant immunity.

KEY MESSAGE: Oomycetes MAMP Pep-13 can trigger SERK3/BAK1-independent PTI. Silencing of SERK3/BAK1 in solanaceous plants resulted in reduced expression of brassinosteroid marker genes and enhanced PTI transcriptional responses to Pep-13 treatment. To prevent disease, pattern recognition receptors (PRRs) are responsible for detecting microbe-associated molecular patterns (MAMPs) to switch on plant innate immunity. SOMATIC EMBROYOGENESIS KINASE 3 (SERK3)/BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) is a well-characterized receptor-like kinase (RLK) that serves as a pivotal co-receptor with PRRs to activate immunity following recognition of MAMPs including flg22, EF-Tu, INF1 and XEG1. However, the requirement for SERK3/BAK1 in many pattern-triggered immune (PTI) signaling pathways is not yet known. Pep-13 is an oomycete MAMP that consists of a highly conserved motif (an oligopeptide of 13 amino acids) shared in Phytophthora transglutaminases. Quantitative RT-PCR analysis reveals that the transcripts of three PTI marker genes (WRKY7, WRKY8 and ACRE31) rapidly accumulate in response to three different MAMPs: flg22, chitin and Pep-13. Whereas silencing of SERK3/BAK1 in Nicotiana benthamiana or potato compromised transcript accumulation in response to flg22, it did not attenuate WRKY7, WRKY8 and ACRE31 up-regulation in response to chitin or Pep-13. This indicates that Pep-13 triggers immunity in a SERK3/BAK1-independent manner, similar to chitin. Surprisingly, silencing of SERK3/BAK1 led to significantly increased accumulation of PTI marker gene transcripts following Pep-13 or chitin treatment, compared to controls. This was accompanied by reduced expression of brassinosteroid (BR) marker genes StSTDH, StEXP8 and StCAB50 and StCHL1, which is a negative regulator of PTI, supporting previous reports that SERK3/BAK1-dependent BR signaling attenuates plant immunity. We provide Pep-13 as an alternative to chitin as a trigger of SERK3/BAK1-independent immunity.

Protein nanocarrier-enabled plant genetic engineering systems.

Genetic engineering can enhance crop yields by developing climate-resilient crop varieties. Nanobiotechnology plays a crucial role in precision delivery of genetic materials, nutrients, and stress-responsive agents into plant cells. This forum highlights recent advances in biodegradable protein-based nanocarrier systems for plant genome editing to transform agricultural practices.

Chloroplast protein StFC-II was manipulated by a Phytophthora effector to enhance host susceptibility.

Oomycete secretes a range of RxLR effectors into host cells to manipulate plant immunity by targeting proteins from several organelles. In this study, we report that chloroplast protein StFC-II is hijacked by a pathogen effector to enhance susceptibility. Phytophthora infestans RxLR effector Pi22922 is activated during the early stages of P. infestans colonization. Stable overexpression of Pi22922 in plants suppresses flg22-triggered reactive oxygen species (ROS) burst and enhances leaf colonization by P. infestans. A potato ferrochelatase 2 (FC-II, a nuclear-encoded chloroplast-targeted protein), a key enzyme for heme biosynthesis in chloroplast, was identified as a target of Pi22922 in the cytoplasm. The pathogenicity of Pi22922 in plants is partially dependent on FC-II. Overexpression of StFC-II decreases resistance of potato and Nicotiana benthamiana against P. infestans, and silencing of NbFC-II in N. benthamiana reduces P. infestans colonization. Overexpression of StFC-II increases heme content and reduces chlorophyll content and photosynthetic efficiency in potato leaves. Moreover, ROS accumulation both in chloroplast and cytoplasm is attenuated and defense-related genes are down-regulated in StFC-II overexpression transgenic potato and N. benthamiana leaves. Pi22922 inhibits E3 ubiquitin ligase StCHIP-mediated StFC-II degradation in the cytoplasm and promotes its accumulation in chloroplasts. In summary, this study characterizes a new mechanism that an oomycete RxLR effector suppresses host defenses by promoting StFC-II accumulation in chloroplasts, thereby compromising the host immunity and promoting susceptibility.

Recent advances in nano-enabled immunomodulation for enhancing plant resilience against phytopathogens.

Plant diseases caused by microbial pathogens pose a severe threat to global food security. Although genetic modifications can improve plant resistance; however, environmentally sustainable strategies are needed to manage plant diseases. Nano-enabled immunomodulation involves using engineered nanomaterials (ENMs) to modulate the innate immune system of plants and enhance their resilience against pathogens. This emerging approach provides unique opportunities through the ability of ENMs to act as nanocarriers for delivering immunomodulatory agents, nanoprobes for monitoring plant immunity, and nanoparticles (NPs) that directly interact with plant cells to trigger immune responses. Recent studies revealed that the application of ENMs as nanoscale agrochemicals can strengthen plant immunity against biotic stress by enhancing systemic resistance pathways, modulating antioxidant defense systems, activating defense-related genetic pathways and reshaping the plant-associated microbiomes. However, key challenges remain in unraveling the complex mechanisms through which ENMs influence plant molecular networks, assessing their long-term environmental impacts, developing biodegradable formulations, and optimizing targeted delivery methods. This review provides a comprehensive investigation of the latest research on nano-enabled immunomodulation strategies, potential mechanisms of action, and highlights future perspectives to overcome existing challenges for sustainable plant disease management.

Modulation of rhizosphere microbial community and metabolites by bio-functionalized nanoscale silicon oxide alleviates cadmium-induced phytotoxicity in bayberry plants.

Cadmium (Cd) is an extremely toxic heavy metal that can originate from industrial activities and accumulate in agricultural soils. This study investigates the potential of biologically synthesized silicon oxide nanoparticles (Bio-SiNPs) in alleviating Cd toxicity in bayberry plants. Bio-SiNPs were synthesized using the bacterial strain Chryseobacterium sp. RTN3 and thoroughly characterized using advanced techniques. A pot experiment results demonstrated that Cd stress substantially reduced leaves biomass, photosynthesis efficiency, antioxidant enzyme activity, and induced oxidative damage in bayberry (Myrica rubra) plants. However, Bio-SiNPs application at 200 mg kg-1 significantly enhanced plant biomass, chlorophyll content (26.4 %), net photosynthetic rate (8.6 %), antioxidant enzyme levels, and mitigated reactive oxygen species production under Cd stress. Bio-SiNPs modulated key stress-related phytohormones by increasing salicylic acid (13.2 %) and abscisic acid (13.7 %) contents in plants. Bio-SiNPs augmented Si deposition on root surfaces, preserving normal ultrastructure in leaf cells. Additionally, 16S rRNA gene sequencing demonstrated that Bio-SiNPs treatment favorably reshaped structure and abundance of specific bacterial groups (Proteobacteria, Actinobacteriota, and Acidobacteriota) in the rhizosphere. Notably, Bio-SiNPs application significantly modulated the key metabolites (phenylacetaldehyde, glycitein, maslinic acid and methylmalonic acid) under both normal and Cd stress conditions. Overall, this study highlights that bio-nanoremediation using Bio-SiNPs enhances tolerance to Cd stress in bayberry plants by beneficially modulating biochemical, microbial, and metabolic attributes.

Fertilization of Microbial Composts: A Technology for Improving Stress Resilience in Plants.

Microbial compost plays a crucial role in improving soil health, soil fertility, and plant biomass. These biofertilizers, based on microorganisms, offer numerous benefits such as enhanced nutrient acquisition (N, P, and K), production of hydrogen cyanide (HCN), and control of pathogens through induced systematic resistance. Additionally, they promote the production of phytohormones, siderophore, vitamins, protective enzymes, and antibiotics, further contributing to soil sustainability and optimal agricultural productivity. The escalating generation of organic waste from farm operations poses significant threats to the environment and soil fertility. Simultaneously, the excessive utilization of chemical fertilizers to achieve high crop yields results in detrimental impacts on soil structure and fertility. To address these challenges, a sustainable agriculture system that ensures enhanced soil fertility and minimal ecological impact is imperative. Microbial composts, developed by incorporating characterized plant-growth-promoting bacteria or fungal strains into compost derived from agricultural waste, offer a promising solution. These biofertilizers, with selected microbial strains capable of thriving in compost, offer an eco-friendly, cost-effective, and sustainable alternative for agricultural practices. In this review article, we explore the potential of microbial composts as a viable strategy for improving plant growth and environmental safety. By harnessing the benefits of microorganisms in compost, we can pave the way for sustainable agriculture and foster a healthier relationship between soil, plants, and the environment.

Potato StLecRK-IV.1 negatively regulates late blight resistance by affecting the stability of a positive regulator StTET8.

Plant receptor-like kinases (RLKs) regulate many processes in plants. Many RLKs perform significant roles in plant immunity. Lectin receptor-like kinases (LecRLKs) are a large family of RLKs. However, the function of most of LecRLKs is poorly understood. In this study, we show that a potato LecRLK, StLecRK-IV.1, is involved in plant immunity against Phytophthora infestans. As a negative regulator of immunity, StLecRK-IV.1 is down-regulated by P. infestans and activated by abscisic acid (ABA). The transient expression of StLecRK-IV.1 in Nicotiana benthamiana enhanced P. infestans leaf colonization significantly. In contrast, the disease lesion size caused by P. infestans was reduced in Virus-induced gene silencing (VIGS) of StLecRK-IV.1 orthologue in N. benthamiana, NbLecRK-IV.1, as well as in potato plants with stable RNA interference of StLecRK-IV.1. Tetraspanin-8 (StTET8) was identified to be interacting with StLecRK-IV.1 using a membrane yeast-2-hybrid system, which was further verified by co-immunoprecipitation, a luciferase complementation assay, and a bimolecular fluorescence complementary (BiFC) test. StTET8 is a positive immune regulator that restrains P. infestans infection. The co-expression of StLecRK-IV.1 with StTET8 antagonized the positive roles of StTET8 against P. infestans. Moreover, the co-expression of StTET8 with StLecRK-IV.1 affected the stability of StTET8, which was confirmed by a Western blot assay and confocal assay. Taken together, our work firstly revealed that a potato L-type Lectin RLK, StLecRK-IV.1, negatively regulates plant immunity by targeting a positive regulator, StTET8, through affecting its stability.

Heterologous overexpression of StERF3 triggers cell death in Nicotiana benthamiana.

Programmed cell death plays a crucial role in plant development and disease defense. Here, we report that the expression of StERF3, a potato EAR motif-containing transcription factor, promotes Phytophthora infestans colonization in Nicotiana benthamiana. Transient overexpression of StERF3 induces cell death in N. benthamiana leaves. The substitution of two key amino acids (14th and 19th) in its ERF domain (the DNA binding domain) dramatically altered its cell death-inducing ability. In addition, StERF3△EAR EAR motif-deletion or StERF3AAA mutation abolished the cell death-inducing ability. StERF3 interacted with the co-repressors Topless-related protein 1 (StTPL1) and Topless-related protein 3 (StTPL3) via the EAR motif. Moreover, cell death induced by StERF3 was facilitated by co-expression with StTPL1 or StTPL3. Virus-induced gene silencing (VIGS) of NbTPL1 and NbTPL3 in N. benthamiana compromised the cell death-inducing ability of StERF3. Furthermore, StERF3-induced cell death accompanied with ROS bursts and the upregulation of the respiratory burst oxidase homolog (Rboh) genes NbRbohA and NbRbohC. In addition, several cell death regulator genes, including NbCRTD, NbNCBP, and NbBCPL, and a hypersensitive cell death marker gene Hin1 were upregulated. StERF3 may positively regulate cell death through its EAR motif-mediated transcriptional repressor activity by inhibiting the expression of genes potentially coding the repressor of cell death (CD).

PAMP-responsive ATL gene StRFP1 and its orthologue NbATL60 positively regulate Phytophthora infestans resistance in potato and Nicotiana benthamiana.

Ubiquitination is a post-translational modification that plays a crucial role during the regulation of plant immune signalling. The plant ATL family consists of a large number of putative RING type ubiquitin ligases. We show that potato ATL family gene StRFP1 and its orthologue NbATL60 from N. benthamiana both respond to Phytophthora infestans culture filtrate (CF) and flg22 induction. StRFP1 positively regulates immunity against P. infestans in potato. Ectopic transient expression of StRFP1 or expression of NbATL60 in N. benthamiana also enhances late blight resistance. By contrast, silencing NbATL60 in N. benthamiana reduces late blight resistance and leads to plant growth inhibition. Both StRFP1 and NbATL60 localize to the plasma membrane and intracellular puncta and possess E3 Ligase activity in vitro. Furthermore we demonstrate that the RING finger domain mutants of StRFP1 and NbATL60 lost E3 ligase activity and fail to suppress P. infestans colonization in N. benthamiana, indicating that E3 ligase activity is critical for StRFP1 and NbATL60 to regulate immunity. Overexpression or RNA interference of StRFP1 in transgenic potato led to increased or decreased expression of PTI maker genes (WRKY7, WRKY8, ACRE31 and Pti5) respectively. Similarly silencing of NbATL60 in N. benthamiana decreases expression of these PTI marker genes. Moreover, VIGS of NbATL60 in N. benthamiana did not compromise P. infestans PAMP INF1 or R2/Avr2, R3a/AVR3a, Rx/Cp and Pto/AvrPto triggered cell death. These results indicate that ATL genes StRFP1 and NbATL60 contribute to basal immunity (PTI) in Solanaceous plants.