Effector-triggered immunity (ETI) is central in plant defense, but whether all cell types execute ETI similarly remains unknown. We combined chemically imposed immune activation with single-cell transcriptomics to profile ETI responses across major leaf cell types in Arabidopsis. Despite uniform ETI perception, we find divergent transcriptional outputs: a core set of defense genes is broadly induced, while distinct cell types activate specialized immune modules. We infer that immune outputs are shaped not only by immune receptor activation but also by cell identity, transcription factor availability, and chromatin accessibility. We further demonstrate that epidermis-enriched transcriptional regulators are required to restrict invasion by non-adapted pathogens. Their absence permits pathogen entry into deeper tissues despite intact recognition, revealing a spatial division of immune functions. Our findings uncover a layered immune architecture in plants, challenge the assumption of uniform immune activation, and establish a framework for exploring cell-type-specific resistance logic in multicellular hosts.

The triploid block leads to seed abortion in crosses involving tetraploid Col-0 pollen. The genetic basis underlying this phenomenon is established in the endosperm and attributed to parental genomic imprinting. This research utilised the genetic variation in Arabidopsis to identify the genomic regions harbouring the maternal modifiers of the triploid block to produce viable large seeds. Distinct chromosomal regions were identified in Bla-1 and Tsu-0 accessions. The Bla-1 maternal modifier maps to the TTG2 locus at the lower end of chromosome 2 to produce large viable seeds in response to a triploid block. Tsu-0 accession, on the other hand, recruits the TTG1 locus on the upper arm of chromosome 5 as a maternal modifier of the triploid block. TTG1 and TTG2 mutations significantly increased the proportion of large viable seeds in interploidy crosses. Both genes are involved in transcriptional regulation in the flavonoid biosynthesis pathway. However, to regulate seed size in diploids, TTG1 functions synergistically with auxin but does so independently of TTG2. This work contributed to the genetic framework for the TTG1 and TTG2 seed size roles.

Background Brassica species is the second most important edible oilseed crop in India. Albugo candida (Pers.) Kuntze, a major oomycete disease of oilseed brassica causing white rust, leads to 60% yield loss globally. The prevalence of A. candida race 2 (Ac2V) that specifically infects B. juncea , coupled with limitations of conventional methods has resulted in a dearth of white rust resistance resources in cultivated varieties. Methods and results In an effort to develop resistant plants, Agrobacterium mediated genetic transformation of three B. juncea genotypes viz., susceptible host var. Varuna, along with its doubled haploid mutant lines C66 and C69 (showing moderate tolerance to field isolates of A. candida ) was initiated to transfer resistance genes ( WRR8 Sf-2 and WRR9 Hi-0 ) identified in Arabidopsis thaliana against race Ac2V, that encode for Toll-like/interleukin-1 receptor-nucleotide binding-leucine-rich repeat proteins that recognize effectors of the pathogen races. Conclusions Our results demonstrate that introduction of resistance genes from a tertiary gene pool by genetic transformation enhances disease resistance in B. juncea genotypes to a highly virulent Ac2V isolate.

Arabidopsis BAK1/SERK3, a co-receptor of leucine-rich repeat pattern recognition receptors (PRRs), mediates pattern-triggered immunity (PTI). Genetic inactivation of BAK1 or BAK1-interacting receptor-like kinases (BIRs) causes cell death, but the direct mechanisms leading to such deregulation remains unclear. Here, we found that the TIR-NBS-LRR protein CONSTITUTIVE SHADE AVOIDANCE 1 (CSA1) physically interacts with BIR3, but not with BAK1. CSA1 mediates cell death in bak1-4 and bak1-4 bir3-2 mutants via components of effector-triggered immunity-(ETI) pathways. Effector HopB1-mediated perturbation of BAK1 also results in CSA1-dependent cell death. Likewise, microbial pattern pg23-induced cell death, but not PTI responses, requires CSA1. Thus, we show that CSA1 guards BIR3 BAK1 homeostasis and integrates pattern- and effector-mediated cell death pathways downstream of BAK1. De-repression of CSA1 in the absence of intact BAK1 and BIR3 triggers ETI cell death. This suggests that PTI and ETI pathways are activated downstream of BAK1 for efficient plant immunity.

Arabidopsis Col-0 RPP2A and RPP2B confer recognition of Arabidopsis downy mildew (Hyaloperonospora arabidopsidis [Hpa]) isolate Cala2, but the identity of the recognized ATR2Cala2 effector was unknown. To reveal ATR2Cala2, an F2 population was generated from a cross between Hpa-Cala2 and Hpa-Noks1. We identified ATR2Cala2 as a non-canonical RxLR-type effector that carries a signal peptide, a dEER motif, and WY domains but no RxLR motif. Recognition of ATR2Cala2 and its effector function were verified by biolistic bombardment, ectopic expression and Hpa infection. ATR2Cala2 is recognized in accession Col-0 but not in Ler-0 in which RPP2A and RPP2B are absent. In ATR2Emoy2 and ATR2Noks1 alleles, a frameshift results in an early stop codon. RPP2A and RPP2B are essential for the recognition of ATR2Cala2. Stable and transient expression of ATR2Cala2 under 35S promoter in Arabidopsis and Nicotiana benthamiana enhances disease susceptibility. Two additional Col-0 TIR-NLR (TNL) genes (RPP2C and RPP2D) adjacent to RPP2A and RPP2B are quantitatively required for full resistance to Hpa-Cala2. We compared RPP2 haplotypes in multiple Arabidopsis accessions and showed that all four genes are present in all ATR2Cala2-recognizing accessions.

The downy mildew oomycete Hyaloperonospora arabidopsidis , an obligate filamentous pathogen, infects Arabidopsis by forming feeding structures called haustoria inside host cells. Previous transcriptome analyses revealed host genes are specifically induced during infection; however, RNA profiling from infected tissues may fail to capture key transcriptional events occurring exclusively in haustoriated host cells where the pathogen injects virulence effectors to modulate host immunity. To determine interactions between Arabidopsis and H. arabidopsidis at the cellular level, we devised a new translating ribosome affinity purification system applicable to inducible, including pathogen-responsive, promoters thus enabling haustoriated cell-specific RNA profiling. Among the host genes specifically expressed in H. arabidopsidis -haustoriated cells, we found genes that promote either susceptibility or resistance to the pathogen, providing new insights into the Arabidopsis/downy mildew interaction. We propose that our novel protocol for profiling cell-specific transcripts will be applicable to several stimulus-specific contexts and other plant-pathogen interactions.

6 1 State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei University, Wuhan 7 430062, China 8 2 The Sainsbury Laboratory, Norwich Research Park, Norwich, NR5 7UH, UK 9 Department of Biological Sciences, School of Science and the Environment, University of 10 Worcester, Worcester, WR2 6AJ, UK 11 The Milner Centre for Evolution, Department of Life Sciences, University of Bath, Bath, 12 BA2 7AY, UK 13

A translating ribosome affinity purification system is used to analyze gene expression in pathogen-infected host cells and identify host genes whose expression is induced in infected cells. The downy mildew oomycete Hyaloperonospora arabidopsidis, an obligate filamentous pathogen, infects Arabidopsis (Arabidopsis thaliana) by forming structures called haustoria inside host cells. Previous transcriptome analyses have revealed that host genes are specifically induced during infection; however, RNA profiling from whole-infected tissues may fail to capture key transcriptional events occurring exclusively in haustoriated host cells, where the pathogen injects virulence effectors to modulate host immunity. To determine interactions between Arabidopsis and H. arabidopsidis at the cellular level, we devised a translating ribosome affinity purification system using 2 high-affinity binding proteins, colicin E9 and Im9 (immunity protein of colicin E9), applicable to pathogen-responsive promoters, thus enabling haustoriated cell-specific RNA profiling. Among the host genes specifically expressed in H. arabidopsidis-haustoriated cells, we found genes that promote either susceptibility or resistance to the pathogen, providing insights into the Arabidopsis-downy mildew interaction. We propose that our protocol for profiling cell-specific transcripts will apply to several stimulus-specific contexts and other plant-pathogen interactions.

SummaryThe oomyceteAlbugo candidacauses white blister rust, an important disease of Brassica crops. Distinct races ofA. candidaare defined by their specificity for infecting different host species.TheWhite Rust Resistance 4(WRR4) locus in Col-0 accession ofArabidopsis thalianacontains three genes that encode TIR-NLR resistance proteins. The Col-0 alleles ofWRR4AandWRR4Bconfer resistance to at least fourA. candidaraces (2, 7 and 9 fromB. juncea, B. rapaandB. oleracea, respectively, and Race 4 fromCapsella bursa-pastoris). Resistance mediated by both paralogs can be overcome by Col-0-virulent isolates of Race 4.After comparing repertoires of candidate effectors in resisted and resistance-breaking strains, we used transient co-expression in tobacco orArabidopsisto identify effectors recognized byWRR4AandWRR4B. A library of CCG effectors from fourA. candidaraces was screened forWRR4A-orWRR4B-dependent elicitation of hypersensitive response (HR). These CCG genes were validated for WRR-dependent HR by bombardment assays in wild type Col-0,wrr4Aorwrr4Bmutants.Our analysis revealed eightWRR4A-recognized CCGs and fourWRR4B-recognized CCGs. Remarkably, the N-terminal region of 100 amino acids after the secretion signal is sufficient forWRR4Arecognition of these eight recognized effectors. This multiple recognition capacity potentially explains the broad-spectrum resistance to manyA. candidaraces conferred byWRR4paralogs.

Albugo candida is an obligate oomycete pathogen that infects many plants in the Brassicaceae family. We resequenced the genome of isolate Ac2V using PacBio long reads and constructed an assembly augmented by Illumina reads. The Ac2VPB genome assembly is 10% larger and more contiguous compared with a previous version. Our annotation of the new assembly, aided by RNA-sequencing information, revealed a 175% expansion (40 to 110) in the CHxC effector class, which we redefined as "CCG" based on motif analysis. This class of effectors consist of arrays of phylogenetically related paralogs residing in gene sparse regions, and shows signatures of positive selection and presence/absence polymorphism. This work provides a resource that allows the dissection of the genomic components underlying A. candida adaptation and, particularly, the role of CCG effectors in virulence and avirulence on different hosts.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

White blister rust, caused by the oomycete Albugo candida , is a widespread disease of Brassica crops. The Arabidopsis CSA1/DAR4 (also known as CSA1/CHS3) paired immune receptor carries an Integrated Domain (ID) with homology to the DA1 family of peptidases. Using domain swaps with DA1 family members, we show that the DAR4 ID acts as an integrated decoy for DAR3, which interacts with and inhibits the peptidase activities of DA1, DAR1 and DAR2 family members. Albugo infection rapidly lowered DAR3 levels and activates DA1 peptidase activity. This promotes endoreduplication of host tissues to support pathogen growth. We propose that DAR4/CSA1 senses the actions of a putative Albugo effector that reduces DAR3 levels and initiates defense.

Nucleotide-binding and leucine-rich repeat receptors (NLRs) are intracellular plant immune receptors that recognize pathogen effectors secreted into the plant cell. Canonical NLRs typically contain three conserved domains including a central nucleotide binding (NB-ARC) domain, C-terminal leucine-rich repeats (LRRs) and an N-terminal domain. A subfamily of plant NLRs contain additional noncanonical domain(s) that have potentially evolved from the integration of the effector targets in the canonical NLR structure. These NLRs with extra domains are thus referred to as NLRs with integrated domains (NLR-IDs). Here, we first summarize our current understanding of NLR-ID activation upon effector binding, focusing on the NLR pairs Pik-1/Pik-2, RGA4/RGA5, and RRS1/RPS4. We speculate on their potential oligomerization into resistosomes as it was recently shown for certain canonical plant NLRs. Furthermore, we discuss how our growing understanding of the mode of action of NLR-ID continuously informs engineering approaches to design new resistance specificities in the context of rapidly evolving pathogens.

SummaryThe oomyceteAlbugo candidacauses white rust of Brassicaceae, including vegetable and oilseed crops, and wild relatives such asArabidopsis thaliana. NovelWhite Rust Resistance(WRR)-genes from Arabidopsis enable new insights into plant/parasite co-evolution.WRR4Afrom Arabidopsis accession Col-0 provides resistance to many but not all white rust races, and encodes a nucleotide-binding (NB), leucine-rich repeat (LRR) (NLR) immune receptor protein. Col-0WRR4Aresistance is broken by a Col-0-virulent isolate ofA. candidarace 4 (AcEx1). We identified an allele ofWRR4Ain Arabidopsis accession Oy-0 and other accessions that confers full resistance to AcEx1.WRR4AOy-0carries a C-terminal extension required for recognition of AcEx1, but reduces recognition of several effectors recognized by theWRR4ACol-0allele.WRR4AOy-0confers full resistance to AcEx1 when expressed as a transgene in the oilseed cropCamelina sativa.SignificanceA C-terminal extension in an allele of the Arabidopsis resistance-protein WRR4A changes effector recognition specificity, enabling theWRR4AOy-0allele to confer immunity toAlbugo candidaraces that overcome theWRR4ACol-0allele. This resistance can be transferred to the oil-producing cropCamelina sativa.Graphical abstract

AbstractThe BRI1-associated kinase BAK1/SERK3 is a positive regulator of multiple leucine rich receptor kinase-mediated signaling pathways including pattern triggered immunity (PTI). Absence or overexpression of BAK1 leads to spontaneous cell death formation. BAK1-interacting receptors (BIR) constitutively interact with BAK1, and plants lacking or overexpressing BIR proteins phenocopy the cell death symptoms observed inbak1knock outs or overexpressors. In the interactome of BIR3, the TIR-NBS-LRR protein CONSTITUTIVE SHADE-AVOIDANCE 1 (CSA1) was identified by mass spectrometry. CSA1 physically interacts with BIR proteins and can be detected in complexes with BAK1. Direct interaction was shown only for CSA1 with BIR proteins but not BAK1. Double mutantbak1 bir3genotypes develop strong dwarfism and cell death symptoms that are dependent on EDS1 and salicylic acid. Loss of CSA1 blocksbak1andbak1 bir3-mediated cell death formation thus demonstrating that CSA1 is causal for this type of cell death. We propose that CSA1 guards BIR proteins and initiates autoimmune cell death that is observed when BAK1 BIR complexes are impaired. Our findings reveal how cell death in the absence of BAK1 and BIR3 is executed and links BAK1, a common co-receptor of many pattern recognition receptors, to NLR proteins typically implicated in effector-triggered immunity.

The BRI1-associated kinase BAK1/SERK3 is a positive regulator of multiple leucine rich receptor kinase-mediated signaling pathways including pattern triggered immunity (PTI). Absence or overexpression of BAK1 leads to spontaneous cell death formation. BAK1-interacting receptors (BIR) constitutively interact with BAK1, and plants lacking or overexpressing BIR proteins phenocopy the cell death symptoms observed in bak1 knock outs or overexpressors. In the interactome of BIR3, the TIR-NBS-LRR protein CONSTITUTIVE SHADE-AVOIDANCE 1 (CSA1) was identified by mass spectrometry. CSA1 physically interacts with BIR proteins and can be detected in complexes with BAK1. Direct interaction was shown only for CSA1 with BIR proteins but not BAK1. Double mutant bak1 bir3 genotypes develop strong dwarfism and cell death symptoms that are dependent on EDS1 and salicylic acid. Loss of CSA1 blocks bak1 and bak1 bir3 -mediated cell death formation thus demonstrating that CSA1 is causal for this type of cell death. We propose that CSA1 guards BIR proteins and initiates autoimmune cell death that is observed when BAK1 BIR complexes are impaired. Our findings reveal how cell death in the absence of BAK1 and BIR3 is executed and links BAK1, a common co-receptor of many pattern recognition receptors, to NLR proteins typically implicated in effector-triggered immunity.### Competing Interest StatementThe authors have declared no competing interest.

Crop production in agriculture is affected by plant genetic background and environmental factors. The application of modern molecular biology tools to conventional plant breeding approaches has facilitated the plant genetic improvement attempts. After the extensive employment of recombinant DNA technology in diverse plant species and despite achievements, the use of transgenic crops has been encountered with public concerns due to the presence of transgenes. The advent of sequence-specific nuclease-based editing technologies especially clustered regularly interspaced short palindromic repeat-associated protein system (CRISPR/Cas) has opened a promising avenue in genetic engineering of plants. The importance of this approach is emphasized since it is a simple and robust tool, moreover, non-transgenic mutants can be selected in later generations. Following the successful use of the CRISPR/Cas9 editing tool in model plants, the applications of this system have been increasingly reported in different plant species. This chapter reviews the contribution of the CRISPR/Cas9 system in the development of genetically modified crops with improved yield, nutritional value, and response to biotic and abiotic stress factors.

Environmental factors along with plant genetic background affect crop productivity. Crop losses are caused by biotic agents including insect pests and pathogens, while others could be due to abiotic factors. However, a significant part of the losses occurs during the transportation, storage and marketing of crops that bring additional costs after harvest and when they reach consumers. Traditional methods have been applied to improve crop quality and reduce postharvest losses. The need to provide adequate food for the growing population, however, leaves the conventional methods insufficient. Genetic engineering of plants overcomes obstacles existing in classical plant breeding methods and thus played a pivotal role in the production of products with the desired properties in modern agriculture. Genome modifications toward sustainably delivering sufficient nutrients to the populations suffering from limited food resources as well as reducing the pre- and postharvest losses have been approved as a promising approach. This chapter reviews the current state of the transgenic fruits and vegetables developed for better nutritional quality and shelf life, and assess the impact of modern biotechnological tools in revolutionizing agriculture.

Arabidopsis thaliana accessions are universally resistant at the adult leaf stage to white rust (Albugo candida) races that infect the crop species Brassica juncea and Brassica oleracea. We used transgressive segregation in recombinant inbred lines to test if this apparent species-wide (nonhost) resistance in A. thaliana is due to natural pyramiding of multiple Resistance (R) genes. We screened 593 inbred lines from an Arabidopsis multiparent advanced generation intercross (MAGIC) mapping population, derived from 19 resistant parental accessions, and identified two transgressive segregants that are susceptible to the pathogen. These were crossed to each MAGIC parent, and analysis of resulting F2 progeny followed by positional cloning showed that resistance to an isolate of A. candida race 2 (Ac2V) can be explained in each accession by at least one of four genes encoding nucleotide-binding, leucine-rich repeat (NLR) immune receptors. An additional gene was identified that confers resistance to an isolate of A. candida race 9 (AcBoT) that infects B. oleracea. Thus, effector-triggered immunity conferred by distinct NLR-encoding genes in multiple A. thaliana accessions provides species-wide resistance to these crop pathogens.

Most land plant genomes carry genes that encode RPW8-NLR Resistance (R) proteins. Angiosperms carry two RPW8-NLR subclasses: ADR1 and NRG1. ADR1s act as 'helper' NLRs for multiple TIR- and CC-NLR R proteins in Arabidopsis. In angiosperm families, NRG1 co-occurs with TIR-NLR Resistance (R) genes. We tested whether NRG1 is required for signalling of multiple TIR-NLRs. Using CRISPR mutagenesis, we obtained an nrg1a-nrg1b double mutant in two Arabidopsis accessions, and an nrg1 mutant in Nicotiana benthamiana. These mutants are compromised in signalling of all TIR-NLRs tested, including WRR4A, WRR4B, RPP1, RPP2, RPP4 and the pairs RRS1/RPS4, RRS1B/RPS4B, CHS1/SOC3 and CHS3/CSA1. In Arabidopsis, NRG1 is required for the hypersensitive cell death response (HR) and full oomycete resistance, but not for salicylic acid induction or bacterial resistance. By contrast, nrg1 loss of function does not compromise the CC-NLR R proteins RPS5 and MLA. RPM1 and RPS2 (CC-NLRs) function is slightly compromised in an nrg1 mutant. Thus, NRG1 is required for full TIR-NLR function and contributes to the signalling of some CC-NLRs. Some NRG1-dependent R proteins also signal partially via the NRG1 sister clade, ADR1. We propose that some NLRs signal via NRG1 only, some via ADR1 only and some via both or neither.

Physiological races of the oomycete Albugo candida are biotrophic pathogens of diverse plant species, primarily the Brassicaceae, and cause infections that suppress host immunity to other pathogens. However, A.candida race diversity and the consequences of host immunosuppression are poorly understood in the field. We report a method that enables sequencing of DNA of plant pathogens and plant-associated microbes directly from field samples (Pathogen Enrichment Sequencing: PenSeq). We apply this method to explore race diversity in A.candida and to detect A.candida-associated microbes in the field (91 A.candida-infected plants). We show with unprecedented resolution that each host plant species supports colonization by one of 17 distinct phylogenetic lineages, each with an unique repertoire of effector candidate alleles. These data reveal the crucial role of sexual and asexual reproduction, polyploidy and host domestication in A.candida specialization on distinct plant species. Our bait design also enabled phylogenetic assignment of DNA sequences from bacteria and fungi from plants in the field. This paper shows that targeted sequencing has a great potential for the study of pathogen populations while they are colonizing their hosts. This method could be applied to other microbes, especially to those that cannot be cultured.