Genome-Wide Identification and Multi-Stress Response Analysis of the DABB-Type Protein-Encoding Genes in Brassica napus.

The DABB proteins, which are characterized by stress-responsive dimeric A/B barrel domains, have multiple functions in plant biology. In Arabidopsis thaliana, these proteins play a crucial role in defending against various pathogenic fungi. However, the specific roles of DABB proteins in Brassica napus remain elusive. In this study, 16 DABB encoding genes were identified, distributed across 10 chromosomes of the B. napus genome, which were classified into 5 branches based on phylogenetic analysis. Genes within the same branch exhibited similar structural domains, conserved motifs, and three-dimensional structures, indicative of the conservation of BnaDABB genes (BnaDABBs). Furthermore, the enrichment of numerous cis-acting elements in hormone induction and light response were revealed in the promoters of BnaDABBs. Expression pattern analysis demonstrated the involvement of BnaDABBs, not only in the organ development of B. napus but also in response to abiotic stresses and Sclerotinia sclerotiorum infection. Altogether, these findings imply the significant impacts of BnaDABBs on plant growth and development, as well as stress responses.

Regulation of Grain Chalkiness and Starch Metabolism by FLO2 Interaction Factor 3, a bHLH Transcription Factor in Oryza sativa.

Chalkiness is a key determinant that directly affects the appearance and cooking quality of rice grains. Previously, Floury endosperm 2 (FLO2) was reported to be involved in the formation of rice chalkiness; however, its regulation mechanism is still unclear. Here, FLO2 interaction factor 3 (OsFIF3), a bHLH transcription factor, was identified and analyzed in Oryza sativa. A significant increase in chalkiness was observed in OsFIF3-overexpressed grains, coupled with a round, hollow filling of starch granules and reduced grain weight. OsFIF3 is evolutionarily conserved in monocotyledons, but variable in dicotyledons. Subcellular localization revealed the predominant localization of OsFIF3 in the nucleus. The DAP-seq (DNA affinity purification sequencing) results showed that OsFIF3 could affect the transcriptional accumulation of ß-amylase 1, α-amylase isozyme 2A-like, pectinesterase 11, ß-glucosidase 28 like, pectinesterase, sucrose transport protein 1 (SUT1), and FLO2 through the binding of the CACGTG motif on their promoters. Moreover, FLO2 and SUT1 with abundant OsFIF3 binding signals showed significant expression reduction in OsFIF3 overexpression lines, further confirming OsFIF3's role in starch metabolism regulation and energy material allocation. Taken together, these findings show that the overexpression of OsFIF3 inhibits the expression of FLO2 and SUT1, thereby increasing grain chalkiness and affecting grain weight.

SsCak1 Regulates Growth and Pathogenicity in Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a devastating fungal pathogen that causes severe crop losses worldwide. It is of vital importance to understand its pathogenic mechanism for disease control. Through a forward genetic screen combined with next-generation sequencing, a putative protein kinase, SsCak1, was found to be involved in the growth and pathogenicity of S. sclerotiorum. Knockout and complementation experiments confirmed that deletions in SsCak1 caused defects in mycelium and sclerotia development, as well as appressoria formation and host penetration, leading to complete loss of virulence. These findings suggest that SsCak1 is essential for the growth, development, and pathogenicity of S. sclerotiorum. Therefore, SsCak1 could serve as a potential target for the control of S. sclerotiorum infection through host-induced gene silencing (HIGS), which could increase crop resistance to the pathogen.

Epigenetic regulation of N-hydroxypipecolic acid biosynthesis by the AIPP3-PHD2-CPL2 complex.

N-Hydroxypipecolic acid (NHP) is a signaling molecule crucial for systemic acquired resistance (SAR), a systemic immune response in plants that provides long-lasting and broad-spectrum protection against secondary pathogen infections. To identify negative regulators of NHP biosynthesis, we performed a forward genetic screen to search for mutants with elevated expression of the NHP biosynthesis gene FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1). Analysis of two constitutive expression of FMO1 (cef) and one induced expression of FMO1 (ief) mutants revealed that the AIPP3-PHD2-CPL2 protein complex, which is involved in the recognition of the histone modification H3K27me3 and transcriptional repression, contributes to the negative regulation of FMO1 expression and NHP biosynthesis. Our study suggests that epigenetic regulation plays a crucial role in controlling FMO1 expression and NHP levels in plants.

A Forward Genetic Screen in Sclerotinia sclerotiorum Revealed the Transcriptional Regulation of Its Sclerotial Melanization Pathway.

Most plant fungal pathogens that cause worldwide crop losses are understudied, due to various technical challenges. With the increasing availability of sequenced whole genomes of these non-model fungi, effective genetic analysis methods are highly desirable. Here, we describe a newly developed pipeline, which combines forward genetic screening with high-throughput next-generation sequencing to enable quick gene discovery. We applied this pipeline in the notorious soilborne phytopathogen Sclerotinia sclerotiorum and identified 32 mutants with various developmental and growth deficiencies. Detailed molecular studies of three melanization-deficient mutants provide a proof of concept for the effectiveness of our method. A master transcription factor was found to regulate melanization of sclerotia through the DHN (1,8-dihydroxynaphthalene) melanin biosynthesis pathway. In addition, these mutants revealed that sclerotial melanization is important for sclerotia survival under abiotic stresses, sclerotial surface structure, and sexual reproduction. Foreseeably, this pipeline can be applied to facilitate efficient in-depth studies of other non-model fungal species in the future.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

An Amidase Contributes to Full Virulence of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is one of the most notorious and ubiquitous soilborne plant pathogens, causing serious economic losses to a large number of hosts worldwide. Although virulence factors have been identified in this filamentous fungus, including various cell-wall-degrading enzymes, toxins, oxalic acids and effectors, our understanding of its virulence strategies is far from complete. To explore novel factors contributing to disease, a new pipeline combining forward genetic screening and next-generation sequencing was utilized in this study. Analysis of a hypovirulent mutant revealed that a mutation in an amidase-encoding gene, Sscle_10g079050, resulted in reduced virulence. This is a first report on the contribution of an amidase to fungal virulence, likely through affecting oxalic acid homeostasis.

Differential regulation of TNL-mediated immune signaling by redundant helper CNLs.

Higher plants utilize nucleotide-binding leucine-rich repeat domain proteins (NLRs) as intracellular immune receptors to recognize pathogen-derived effectors and trigger a robust defense. The Activated Disease Resistance 1 (ADR1) family of coiled-coil NLRs (CNLs) have evolved as helper NLRs that function downstream of many TIR-type sensor NLRs (TNLs). Close homologs of ADR1s form the N REQUIREMENT GENE 1 (NRG1) family in Arabidopsis, the function of which is unclear. Through CRISPR/Cas9 gene editing methods, we discovered that the tandemly repeated NRG1A and NRG1B are functionally redundant and operate downstream of TNLs with differential strengths. Interestingly, ADR1s and NRG1s function in two distinct parallel pathways contributing to TNL-specific immunity. Synergistic effects on basal and TNL-mediated defense were detected among ADR1s and NRG1s. An intact P-loop of NRG1s is not required for mediating signals from sensor TNLs, whereas auto-active NRG1A exhibits autoimmunity. Importantly, NRG1s localize to the cytosol and endomembrane network regardless of the presence of effectors, suggesting a cytosolic activation mechanism. Taken together, different sensor TNLs differentially use two groups of helper NLRs, ADR1s and NRG1s, to transduce downstream defense signals.

Systems model-guided rice yield improvements based on genes controlling source, sink, and flow.

A large number of genes related to source, sink, and flow have been identified after decades of research in plant genetics. Unfortunately, these genes have not been effectively utilized in modern crop breeding. This perspective paper aims to examine the reasons behind such a phenomenon and propose a strategy to resolve this situation. Specifically, we first systematically survey the currently cloned genes related to source, sink, and flow; then we discuss three factors hindering effective application of these identified genes, which include the lack of effective methods to identify limiting or critical steps in a signaling network, the misplacement of emphasis on properties, at the leaf, instead of the whole canopy level, and the non-linear complex interaction between source, sink, and flow. Finally, we propose the development of systems models of source, sink and flow, together with a detailed simulation of interactions between them and their surrounding environments, to guide effective use of the identified elements in modern rice breeding. These systems models will contribute directly to the definition of crop ideotype and also identification of critical features and parameters that limit the yield potential in current cultivars.

Characteristic and inheritance analysis of targeted mutagenesis mediated by genome editing in rice.

The transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) systems are two current genome editing technologies. Here, we compare and analyze the characteristics of the targeted mutations mediated by these two systems, such as efficiency, type, position, time, and genetic patterns. Both the TALEN and CRISPR/Cas9 systems can induce site-specific mutations in T0 rice plants effectively, but CRISPR/Cas9 is more effective. The major mutation type in both systems is the short insertion/deletion(InDel) mutation within 10 base pairs: deletions ranging from 1 to 10 bps are more often in TALEN, and 1bp insertions are more often in CRISPR/Cas9. Moreover, double-strand breaks (DSBs) generated by CRISPR/Cas9 are more precise than TALEN. In addition, DSBs could be repaired by the homologous recombination at a low frequency, causing DNA fragment duplication mutations. In some cases, the DNA fragments between the two close targets are deleted or inverted, and the mutation efficiency does not positively correlatewith the mutation efficiency of each target. Mutagenesis mediated by the TALEN or CRISPR/Cas9 system can occur as early as in transformed callus cells, and less frequently in somatic cells. Consequently, four different mutation types are formed, including homozygous, heterozygous, bi-allelic and chimeric mutations, with bi-allelic mutations having the highest rate and chimeric mutations having the lowest rate. All, except chimeric mutations, can descend stably into the next generation.

The Chromatin Remodeler SPLAYED Negatively Regulates SNC1-Mediated Immunity.

SNC1 (SUPPRESSOR OF NPR1, CONSTITUTIVE 1) is one of a suite of intracellular Arabidopsis NOD-like receptor (NLR) proteins which, upon activation, result in the induction of defense responses. However, the molecular mechanisms underlying NLR activation and the subsequent provocation of immune responses are only partially characterized. To identify negative regulators of NLR-mediated immunity, a forward genetic screen was undertaken to search for enhancers of the dwarf, autoimmune gain-of-function snc1 mutant. To avoid lethality resulting from severe dwarfism, the screen was conducted using mos4 (modifier of snc1, 4) snc1 plants, which display wild-type-like morphology and resistance. M2 progeny were screened for mutant, snc1-enhancing (muse) mutants displaying a reversion to snc1-like phenotypes. The muse9 mos4 snc1 triple mutant was found to exhibit dwarf morphology, elevated expression of the pPR2-GUS defense marker reporter gene and enhanced resistance to the oomycete pathogen Hyaloperonospora arabidopsidis Noco2. Via map-based cloning and Illumina sequencing, it was determined that the muse9 mutation is in the gene encoding the SWI/SNF chromatin remodeler SYD (SPLAYED), and was thus renamed syd-10. The syd-10 single mutant has no observable alteration from wild-type-like resistance, although the syd-4 T-DNA insertion allele displays enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326. Transcription of SNC1 is increased in both syd-4 and syd-10. These data suggest that SYD plays a subtle, specific role in the regulation of SNC1 expression and SNC1-mediated immunity. SYD may work with other proteins at the chromatin level to repress SNC1 transcription; such regulation is important for fine-tuning the expression of NLR-encoding genes to prevent unpropitious autoimmunity.

Regulation of transcription of nucleotide-binding leucine-rich repeat-encoding genes SNC1 and RPP4 via H3K4 trimethylation.

Plant nucleotide-binding leucine-rich repeat (NB-LRR) proteins serve as intracellular sensors to detect pathogen effectors and trigger immune responses. Transcription of the NB-LRR-encoding Resistance (R) genes needs to be tightly controlled to avoid inappropriate defense activation. How the expression of the NB-LRR R genes is regulated is poorly understood. The Arabidopsis (Arabidopsis thaliana) suppressor of npr1-1, constitutive 1 (snc1) mutant carries a gain-of-function mutation in a Toll/Interleukin1 receptor-like (TIR)-NB-LRR-encoding gene, resulting in the constitutive activation of plant defense responses. A snc1 suppressor screen identified modifier of snc1,9 (mos9), which partially suppresses the autoimmune phenotypes of snc1. Positional cloning revealed that MOS9 encodes a plant-specific protein of unknown function. Expression analysis showed that MOS9 is required for the full expression of TIR-NB-LRR protein-encoding RECOGNITION OF PERONOSPORA PARASITICA 4 (RPP4) and SNC1, both of which reside in the RPP4 cluster. Coimmunoprecipitation and mass spectrometry analyses revealed that MOS9 associates with the Set1 class lysine 4 of histone 3 (H3K4) methyltransferase Arabidopsis Trithorax-Related7 (ATXR7). Like MOS9, ATXR7 is also required for the full expression of SNC1 and the autoimmune phenotypes in the snc1 mutant. In atxr7 mutant plants, the expression of RPP4 is similarly reduced, and resistance against Hyaloperonospora arabidopsidis Emwa1 is compromised. Consistent with the attenuated expression of SNC1 and RPP4, trimethylated H3K4 marks are reduced around the promoters of SNC1 and RPP4 in mos9 plants. Our data suggest that MOS9 functions together with ATXR7 to regulate the expression of SNC1 and RPP4 through H3K4 methylation, which plays an important role in fine-tuning their transcription levels and functions in plant defense.

Histone Methyltransferase SsDim5 Regulates Fungal Virulence through H3K9 Trimethylation in Sclerotinia sclerotiorum.

Histone post-translational modification is one of the main mechanisms of epigenetic regulation, which plays a crucial role in the control of gene expression and various biological processes. However, whether or not it affects fungal virulence in Sclerotinia sclerotiorum is not clear. In this study, we identified and cloned the histone methyltransferase Defective in methylation 5 (Dim5) in S. sclerotiorum, which encodes a protein containing a typical SET domain. SsDim5 was found to be dynamically expressed during infection. Knockout experiment demonstrated that deletion of SsDim5 reduced the virulence in Ssdim5-1/Ssdim5-2 mutant strains, accompanied by a significant decrease in H3K9 trimethylation levels. Transcriptomic analysis further revealed the downregulation of genes associated with mycotoxins biosynthesis in SsDim5 deletion mutants. Additionally, the absence of SsDim5 affected the fungus's response to oxidative and osmotic, as well as cellular integrity. Together, our results indicate that the H3K9 methyltransferase SsDim5 is essential for H3K9 trimethylation, regulating fungal virulence throug mycotoxins biosynthesis, and the response to environmental stresses in S. sclerotiorum.

The Sclerotinia sclerotiorum ADP-Ribosylation Factor 6 Plays an Essential Role in Abiotic Stress Response and Fungal Virulence to Host Plants.

The ADP-ribosylation factor 6 (Arf6), as the only member of the Arf family III protein, has been extensively studied for its diverse biological functions in animals. Previously, the Arf6 protein in Magnaporthe oryzae was found to be crucial for endocytosis and polarity establishment during asexual development. However, its role remains unclear in S. sclerotiorum. Here, we identified and characterized SsArf6 in S. sclerotiorum using a reverse genetic approach. Deletion of SsArf6 impaired hyphal growth and development and produced more branches. Interestingly, knockout of SsArf6 resulted in an augmented tolerance of S. sclerotiorum towards oxidative stress, and increased its sensitivity towards osmotic stress, indicative of the different roles of SsArf6 in various stress responses. Simultaneously, SsArf6 deletion led to an elevation in melanin accumulation. Moreover, the appressorium formation was severely impaired, and fungal virulence to host plants was significantly reduced. Overall, our findings demonstrate the essential role of SsArf6 in hyphal development, stress responses, appressorium formation, and fungal virulence to host plants.

Genome-Wide Identification of GYF-Domain Encoding Genes in Three Brassica Species and Their Expression Responding to Sclerotinia sclerotiorum in Brassica napus.

GYF (glycine-tyrosine-phenylalanine)-domain-containing proteins, which were reported to participate in many aspects of biological processes in yeast and animals, are highly conserved adaptor proteins existing in almost all eukaryotes. Our previous study revealed that GYF protein MUSE11/EXA1 is involved in nucleotide-binding leucine-rich repeat (NLR) receptor-mediated defense in Arabidopsis thaliana. However, the GYF-domain encoding homologous genes are still not clear in other plants. Here, we performed genome-wide identification of GYF-domain encoding genes (GYFs) from Brassica napus and its parental species, Brassica rapa and Brassica oleracea. As a result, 26 GYFs of B. napus (BnaGYFs), 11 GYFs of B. rapa (BraGYFs), and 14 GYFs of B. oleracea (BolGYFs) together with 10 A. thaliana (AtGYFs) were identified, respectively. We, then, conducted gene structure, motif, cis-acting elements, duplication, chromosome localization, and phylogenetic analysis of these genes. Gene structure analysis indicated the diversity of the exon numbers of these genes. We found that the defense and stress responsiveness element existed in 23 genes and also identified 10 motifs in these GYF proteins. Chromosome localization exhibited a similar distribution of BnaGYFs with BraGYFs or BolGYFs in their respective genomes. The phylogenetic and gene collinearity analysis showed the evolutionary conservation of GYFs among B. napus and its parental species as well as Arabidopsis. These 61 identified GYF domain proteins can be classified into seven groups according to their sequence similarity. Expression of BnaGYFs induced by Sclerotinia sclerotiorum provided five highly upregulated genes and five highly downregulated genes, which might be candidates for further research of plant-fungal interaction in B. napus.

mRNA Turnover Protein 4 Is Vital for Fungal Pathogenicity and Response to Oxidative Stress in Sclerotinia sclerotiorum.

Ribosome assembly factors have been extensively studied in yeast, and their abnormalities may affect the assembly process of ribosomes and cause severe damage to cells. However, it is not clear whether mRNA turnover protein 4 (MRT4) functions in the fungal growth and pathogenicity in Sclerotinia sclerotiorum. Here, we identified the nucleus-located gene SsMRT4 using reverse genetics, and found that knockdown of SsMRT4 resulted in retard mycelia growth and complete loss of pathogenicity. Furthermore, mrt4 knockdown mutants showed almost no appressorium formation and oxalic acid production comparing to the wild-type and complementary strains. In addition, the abilities to ROS elimination and resistance to oxidative and osmotic stresses were also seriously compromised in mrt4 mutants. Overall, our study clarified the role of SsMRT4 in S. sclerotiorum, providing new insights into ribosome assembly in regulating pathogenicity and resistance to environmental stresses of fungi.

Identification of receptor-like proteins induced by Sclerotinia sclerotiorum in Brassica napus.

Heightening the resistance of plants to microbial infection is a widely concerned issue, especially for economical crops. Receptor-like proteins (RLPs), typically with tandem leucine-rich repeats (LRRs) domain, play a crucial role in mediating immune activation, being an indispensable constituent in the first layer of defense. Based on an analysis of orthologs among Brassica rapa, Brassica oleracea, and Brassica napus using Arabidopsis thaliana RLPs as a reference framework, we found that compared to A. thaliana, there were some obvious evolutionary diversities of RLPs among the three Brassicaceae species. BnRLP encoding genes were unevenly distributed on chromosomes, mainly on chrA01, chrA04, chrC03, chrC04, and chrC06. The orthologs of five AtRLPs (AtRLP3, AtRLP10, AtRLP17, AtRLP44, and AtRLP51) were highly conserved, but retrenchment and functional centralization occurred in Brassicaceae RLPs during evolution. The RLP proteins were clustered into 13 subgroups. Ten BnRLPs presented expression specificity between R and S when elicited by Sclerotinia sclerotiorum, which might be fabulous candidates for S. sclerotiorum resistance research.

A comparative study on photosynthetic characteristics and flavonoid metabolism between Camellia petelotii (Merr.) Sealy and Camellia impressinervis Chang &Liang.

Camellia petelotii (Merr.) Sealy and Camellia impressinervis Chang & Liang belong to the golden subgroup of Camellia (Theaceae). This subgroup contains the yellow-flowering species of the genus, which have high medicinal and ornamental value and a narrow geographical distribution. These species differ in their tolerance to high light intensity. This study aimed to explore the differences in their light-stress responses and light damage repair processes, and the effect of these networks on secondary metabolite synthesis. Two-year-old plants of both species grown at 300 µmol·m-2·s-1 photosynthetically active radiation (PAR) were shifted to 700 µmol·m-2·s-1 PAR for 5 days shifting back to 300 µmol·m-2·s-1 PAR for recovery for 5 days. Leaf samples were collected at the start of the experiment and 2 days after each shift. Data analysis included measuring photosynthetic indicators, differential transcriptome expression, and quantifying plant hormones, pigments, and flavonoids. Camellia impressinervis showed a weak ability to recover from photodamage that occurred at 700 µmol·m-2·s-1 compared with C. petelotii. Photodamage led to decreased photosynthesis, as shown by repressed transcript abundance for photosystem II genes psbA, B, C, O, and Q, photosystem I genes psaB, D, E, H, and N, electron transfer genes petE and F, and ATP synthesis genes ATPF1A and ATPF1B. High-light stress caused more severe damage to C. impressinervis, which showed a stronger response to reactive oxygen species than C. petelotii. In addition, high-light stress promoted the growth and development of high zeatin signalling and increased transcript abundance of adenylate dimethylallyl transferase (IPT) and histidine-containing phosphotransferase (AHP). The identification of transcriptional differences in the regulatory networks that respond to high-light stress and activate recovery of light damage in these two rare species adds to the resources available to conserve them and improve their value through molecular breeding.

Activation of NLR-Mediated Autoimmunity in Arabidopsis Early in Short Days 4 Mutant.

From a reverse genetic screen using CRISPR/Cas9 gene editing tool, we unintentionally identified an autoimmune mutant. Map-based cloning and whole-genome sequencing revealed that it contains a deletion in SMALL UBIQUITIN-RELATED MODIFIER (SUMO) protease encoding gene EARLY IN SHORT DAYS 4 (ESD4). Previous studies reported that esd4 mutants accumulate elevated levels of plant defense hormone salicylic acid (SA). However, upregulated PATHOGENESIS-RELATED GENE 1 (PR1) expression in esd4 only partly relies on SA level. In this study, we show that plant metabolite N-hydroxypipecolic acid (NHP) biosynthetic genes are upregulated in esd4, and NHP biosynthesis mutant flavin-dependent-monooxygenase 1 (fmo1) partially suppresses the autoimmune phenotypes of esd4, suggestive of a requirement of NHP signaling for the autoimmunity in esd4. As activation of nucleotide-binding leucine-rich repeat immune receptors (NLRs) are associates with the biosynthesis of SA and NHP and lipase-like protein ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) is a key component downstream of many NLRs, we examined the relationship between EDS1 and ESD4 by analyzing the eds1 esd4 double mutant. We found that eds1 largely suppresses esd4 autoimmunity and blocks the elevated expressions of SA and NHP biosynthesis-related genes in esd4. Overall, our study provides evidence supporting the hypothesis that SUMO protease ESD4 likely targets a yet to be identified guardee of NLR by removing its SUMO modification to avoid recognition by the cognate NLR. Loss of ESD4 results in activation of NLR-mediated autoimmunity.

SsNEP2 Contributes to the Virulence of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a notorious soilborne fungal pathogen that causes serious economic losses globally. The necrosis and ethylene-inducible peptide 1 (NEP1)-like proteins (NLPs) were previously shown to play an important role in pathogenicity in fungal and oomycete pathogens. Here, we generated S. sclerotiorum necrosis and ethylene-inducible peptide 2 (SsNEP2) deletion mutant through homologous recombination and found that SsNEP2 contributes to the virulence of S. sclerotiorum without affecting the development of mycelia, the formation of appressoria, or the secretion of oxalic acid. Although knocking out SsNEP2 did not affect fungal sensitivity to oxidative stress, it did lead to decreased accumulation of reactive oxygen species (ROS) in S. sclerotiorum. Furthermore, Ssnlp24SsNEP2 peptide derived from SsNEP2 triggered host mitogen-activated protein kinase (MAPK) activation, increased defense marker gene expression, and enhanced resistance to Hyaloperonospora arabidopsidis Noco2. Taken together, our data suggest that SsNEP2 is involved in fungal virulence by affecting ROS levels in S. sclerotiorum. It can serve as a pathogen-associated molecular pattern (PAMP) and trigger host pattern triggered immunity to promote the necrotrophic lifestyle of S. sclerotiorum.

A Single Amino Acid Substitution in RFC4 Leads to Endoduplication and Compromised Resistance to DNA Damage in Arabidopsis thaliana.

Replication factor C (RFC) is a heteropentameric ATPase associated with the diverse cellular activities (AAA+ATPase) protein complex, which is composed of one large subunit, known as RFC1, and four small subunits, RFC2/3/4/5. Among them, RFC1 and RFC3 were previously reported to mediate genomic stability and resistance to pathogens in Arabidopsis. Here, we generated a viable rfc4e (rfc4-1/RFC4G54E) mutant with a single amino acid substitution by site-directed mutagenesis. Three of six positive T2 mutants with the same amino acid substitution, but different insertion loci, were sequenced to identify homozygotes, and the three homozygote mutants showed dwarfism, early flowering, and a partially sterile phenotype. RNA sequencing revealed that genes related to DNA repair and replication were highly upregulated. Moreover, the frequency of DNA lesions was found to be increased in rfc4e mutants. Consistent with this, the rfc4e mutants were very sensitive to DSB-inducing genotoxic agents. In addition, the G54E amino acid substitution in AtRFC4 delayed cell cycle progression and led to endoduplication. Overall, our study provides evidence supporting the notion that RFC4 plays an important role in resistance to genotoxicity and cell proliferation by regulating DNA damage repair in Arabidopsis thaliana.