As a universal process in multicellular organisms, including animals and plants, cells usually emit danger signals when suffering from attacks of microbes and herbivores, or physical damage. These signals, termed as damage-associated molecular patterns (DAMPs), mainly include cell wall or extracellular protein fragments, peptides, nucleotides, and amino acids. Once exposed on cell surfaces, DAMPs are detected by plasma membrane-localized receptors of surrounding cells to regulate immune responses against the invading organisms and promote damage repair. DAMPs may also act as long-distance mobile signals to mediate systemic wounding responses. Generation, release, and perception of DAMPs, and signaling events downstream of DAMP perception are all rigorously modulated by plants. These processes integrate together to determine intricate mechanisms of DAMP-triggered immunity in plants. In this review, we present an extensive overview on our current understanding of DAMPs in plant immune system.

Besides the KU-dependent classical non-homologous end-joining (C-NHEJ) pathway, an alternative NHEJ pathway first identified in mammalian systems, which is often called the back-up NHEJ (B-NHEJ) pathway, was also found in plants. In mammalian systems PARP was found to be one of the essential components in B-NHEJ. Here we investigated whether PARP1 and PARP2 were also involved in B-NHEJ in Arabidopsis. To this end Arabidopsis parp1, parp2 and parp1parp2 (p1p2) mutants were isolated and functionally characterized. The p1p2 double mutant was crossed with the C-NHEJ ku80 mutant resulting in the parp1parp2ku80 (p1p2k80) triple mutant. As expected, because of their role in single strand break repair (SSBR) and base excision repair (BER), the p1p2 and p1p2k80 mutants were shown to be sensitive to treatment with the DNA damaging agent MMS. End-joining assays in cell-free leaf protein extracts of the different mutants using linear DNA substrates with different ends reflecting a variety of double strand breaks were performed. The results showed that compatible 5'-overhangs were accurately joined in all mutants, that KU80 protected the ends preventing the formation of large deletions and that PARP proteins were involved in microhomology mediated end joining (MMEJ), one of the characteristics of B-NHEJ.

Double-strand breaks (DSBs) are one of the most harmful DNA lesions. Cells utilize two main pathways for DSB repair: homologous recombination (HR) and nonhomologous end-joining (NHEJ). NHEJ can be subdivided into the KU-dependent classical NHEJ (c-NHEJ) and the more error-prone KU-independent backup-NHEJ (b-NHEJ) pathways, involving the poly (ADP-ribose) polymerases (PARPs). However, in the absence of these factors, cells still seem able to adequately maintain genome integrity, suggesting the presence of other b-NHEJ repair factors or pathways independent from KU and PARPs. The outcome of DSB repair by NHEJ pathways can be investigated by using artificial sequence-specific nucleases such as CRISPR/Cas9 to induce DSBs at a target of interest. Here, we used CRISPR/Cas9 for DSB induction at the Arabidopsis cruciferin 3 (CRU3) and protoporphyrinogen oxidase (PPO) genes. DSB repair outcomes via NHEJ were analyzed using footprint analysis in wild-type plants and plants deficient in key factors of c-NHEJ (ku80), b-NHEJ (parp1 parp2), or both (ku80 parp1 parp2). We found that larger deletions of >20 bp predominated after DSB repair in ku80 and ku80 parp1 parp2 mutants, corroborating with a role of KU in preventing DSB end resection. Deletion lengths did not significantly differ between ku80 and ku80 parp1 parp2 mutants, suggesting that a KU- and PARP-independent b-NHEJ mechanism becomes active in these mutants. Furthermore, microhomologies and templated insertions were observed at the repair junctions in the wild type and all mutants. Since these characteristics are hallmarks of polymerase θ-mediated DSB repair, we suggest a possible role for this recently discovered polymerase in DSB repair in plants.

Various pathogenic species are capable of penetrating plant leaves through stomata on the leaf surface for propagation by absorbing nutrients in plant interiors. Plants have evolved abilities to close stomata to restrict pathogen infections. The model plant Arabidopsis (Arabidopsis thaliana) closes stomata when FLAGELLIN SENSING2 (FLS2), a receptor protein localized in the plasma membrane (PM) of stomatal guard cells, detects flagellin, a pathogen-associated molecular pattern (PAMP) derived from the bacterial pathogen Pseudomonas syringae. It currently remains largely unknown how flagellin-FLS2 signaling initiates stomatal closure. Our previous studies showed that PAMP-INDUCED PEPTIDE1 (PIP1), an Arabidopsis endogenous peptide, activates immune responses through a PM-localized receptor, RECEPTOR-LIKE KINASE7 (RLK7). Here, we demonstrate that PIP1-RLK7 act downstream of FLS2 to activate stomatal immunity against the bacterial strain Pseudomonas syringe pv. tomato (Pst) DC3118. PIP1 promotes the expression of genes involved in salicylic acid (SA) biosynthesis. SA contributes to the expression of PIP1 preligand prePIP1 and the PIP1-induced stomatal closure. In contrast, methl jasmonate (MJ) and a pathogen-derived jasmonate mimic coronatine (COR) performs an opposite function of SA. SA also promotes the PIP1-induced production of reactive oxygen species (ROS) which is required for PIP1-induced stomatal closure. Overall, PIP1 and SA may form a positive feedback loop to regulate ROSmediated stomatal immunity in Arabidopsis.

Agrobacterium tumefaciens, a pathogenic bacterium capable of transforming plants through horizontal gene transfer, is nowadays the preferred vector for plant genetic engineering. The vehicle for transfer is the T-strand, a single-stranded DNA molecule bound by the bacterial protein VirD2, which guides T-DNA into the plants nucleus where it integrates. How VirD2 is removed from T-DNA, and which mechanism acts to attach the liberated end to the plant genome is currently unknown. Here, using newly developed technology that yields hundreds of T-DNA integrations in somatic tissue of Arabidopsis thaliana, we uncover two redundant mechanisms for the genomic capture of the T-DNA's 5' end. Different from capture of the 3' end of the T-DNA, which is the exclusive action of polymerase theta-mediated end joining (TMEJ), 5' attachment is accomplished either by TMEJ or by canonical non-homologous end joining (cNHEJ). We further nd that TMEJ needs MRE11, whereas cNHEJ requires TDP2 to remove the 5'-end blocking protein VirD2. As a consequence, T-DNA integration is severely impaired in plants de cient for both MRE11 and TDP2 (or other cNHEJ factors). In support of MRE11 and cNHEJ speci cally acting on the 5' end, we demonstrate rescue of the integration defect of double-de cient plants by using T-DNAs that are capable of forming telomeres upon 3' capture. Our study provides a mechanistic model for how Agrobacterium exploits the plant's own DNA repair machineries to transform them. Main TextAgrobacterium tumefaciens-mediated transformation (AMT) is the most widely used method for generating transgenic plants. In nature, this soil bacterium transforms dicotyledonous plants by translocating part of its DNA, the transferred (T)-DNA, into plant cells, where it integrates into the plant's genome 1 . Subsequent expression of the Agrobacterium genes causes crown gall disease. Within Agrobacterium the T-DNA is located on a Tumor-inducing (Ti) plasmid anked by a repeated sequence of 25 bp, the Left and Right Border repeat (LB and RB). These sequences are recognition sites for the virulence proteins VirD1 and VirD2, which generate ssDNA breaks required to liberate T-DNA as a singlestranded DNA molecule, the T-strand 2 . The VirD2 protein remains covalently bound to the 5' end of the Tstrand 3,4 and pilots it into the plant cell through the type 4 secretion system 5 that is created by the Agrobacterium virulence program upon detection of wounded plant cells 6 . The T-DNA is subsequently imported into the nucleus 7 where it integrates at a random position in the genome 8 . The molecular mechanism by which the T-DNA is integrated into the plant genome has remained enigmatic until recently when it was found for Arabidopsis thaliana that this process critically depends on polymerase theta (Pol θ) 9 , a host protein that acts in the repair of DNA double-strand breaks (DSBs) via end joining. Abundant genetic and biochemical research performed over the last few years has established that Pol θ facilitates repair of DSBs in a multitude of species ...

Double-strand breaks (DSBs) are considered to be one of the most harmful and mutagenic forms of DNA damage. They are highly toxic if unrepaired, and can cause genome rearrangements and even cell death. Cells employ two major pathways to repair DSBs: homologous recombination (HR) and non-homologous end-joining (NHEJ). In plants, most applications of genome modification techniques depend on the development of DSB repair pathways, such as Agrobacterium-mediated transformation (AMT) and gene targeting (GT). In this paper, we review the achieved knowledge and recent advances on the DNA DSB response and its main repair pathways; discuss how these pathways affect Agrobacterium-mediated T-DNA integration and gene targeting in plants; and describe promising strategies for producing DSBs artificially, at definite sites in the genome.

Agrobacterium tumefaciens, a pathogenic bacterium capable of transforming plants through horizontal gene transfer, is nowadays the preferred vector for plant genetic engineering. The vehicle for transfer is the T-strand, a single-stranded DNA molecule bound by the bacterial protein VirD2, which guides T-DNA into the plants nucleus where it integrates. How VirD2 is removed from T-DNA, and which mechanism acts to attach the liberated end to the plant genome is currently unknown. Here, using newly developed technology that yields hundreds of T-DNA integrations in somatic tissue of Arabidopsis thaliana, we uncover two redundant mechanisms for the genomic capture of the T-DNA’s 5’ end. Different from capture of the 3’ end of the T-DNA, which is the exclusive action of polymerase theta-mediated end joining (TMEJ), 5’ attachment is accomplished either by TMEJ or by canonical non-homologous end joining (cNHEJ). We further find that TMEJ needs MRE11, whereas cNHEJ requires TDP2 to remove the 5’-end blocking protein VirD2. As a consequence, T-DNA integration is severely impaired in plants deficient for both MRE11 and TDP2 (or other cNHEJ factors). In support of MRE11 and cNHEJ specifically acting on the 5’ end, we demonstrate rescue of the integration defect of double-deficient plants by using T-DNAs that are capable of forming telomeres upon 3’ capture. Our study provides a mechanistic model for how Agrobacterium exploits the plant’s own DNA repair machineries to transform them.