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Plant genomes are characterized by large and complex gene families that often result in similar and partially overlapping functions. This genetic redundancy severely hampers current efforts to uncover novel phenotypes, delaying basic genetic research and breeding programmes. Here we describe the development and validation of Multi-Knock, a genome-scale clustered regularly interspaced short palindromic repeat toolbox that overcomes functional redundancy in Arabidopsisby simultaneously targeting multiple gene-family members, thus identifying genetically hidden components. We computationally designed 59,129 optimal single-guide RNAs that each target two to ten genes within a family at once. Furthermore, partitioning the library into ten sublibraries directed towards a different functional group allows flexible and targeted genetic screens. From the 5,635 single-guide RNAs targeting the plant transportome, we generated over 3,500 independent Arabidopsislines that allowed us to identify and characterize the first known cytokinin tonoplast-localized transporters in plants. With the ability to overcome functional redundancy in plants at the genome-scale level, the developed strategy can be readily deployed by scientists and breeders for basic research and to expedite breeding efforts.

•The fungal elicitor, Xyn11/eix, triggers characteristic defense responses in Prunus persica(peach) and Prunus avium(sweet cherry) species: increased production of ethylene, and induction of cell death.•Functional orthologues of LeEIX2, a known Xyn11/eix receptor in Solanum lycopersicum, were identified in both Prunus persica(PpEIX2–2) and Prunus avium(PaEIX2).•Our findings indicate a similar PTI response for Xyn11/six recognition and signaling across Rosaceae and Solanaceae plant families.

Nanostructured electrodes detecting bacteria or viruses through DNA hybridization represent a promising method, which may be useful in on-field applications where PCR-based methods are very expensive, time-consuming, and require trained personnel. Indeed, electrochemical sensors combine disposability, fast response, high sensitivity, and portability. Here, a low-cost and high-surface-area electrode, based on Au-decorated NiO nanowalls, demonstrates a highly sensitive PCR-free detection of a real sample of Mycoplasma agalactiae(Ma) DNA. NiO nanowalls, synthesized by aqueous methods, thermal annealing, and Au decoration, by electroless deposition, ensure a high-surface-area platform for successful immobilization of Ma thiolated probe DNA. The morphological, chemical, and electrochemical properties of the electrode were characterized, and a reproducible detection of synthetic Ma DNA was observed and investigated by impedance measurements. Electrochemical impedance spectroscopy (EIS) ascribed the origin of impedance signal to the Ma DNA hybridization with its probe immobilized onto the electrode. The electrode successfully discriminates between DNA extracted from healthy and infected sheep milk, showing the ability to detect Ma DNA in concentrations as low as 53 ± 2 copy number μL–1. The Au-decorated NiO nanowall electrode represents a promising route toward PCR-free, disposable, rapid, and molecular detection.

The response of Nicotiana tabacum to tentoxin (chlorosis) is inherited with chloroplasts. N. tabacum var. Xanthi, a tentoxin-resistant line, was used to pollinate tentoxin-sensitive N. tabacum line 92, an alloplasmic male-sterile line containing N. undulata plastids. The seeds were mutagenized with nitrosomethylurea and germinated in the presence of tentoxin. Two percent of the seedlings had green sectors in their first true leaves. These plants were grown to maturity under non-selective conditions. Homogeneous tentoxin-resistant lines were obtained in the third generation. DNA analysis indicated, however, that selection for paternal plastids, rather than mutagenesis of maternal ones, had occurred in the tentoxin-resistant progeny. Mitochondria, which were not under selection pressure, were inherited maternally as expected. Inheritance of tentoxin-resistant paternal plastids did not require seed mutagenesis. Normally germinated seedlings that were kept under tentoxin selection consistently produced a low level of resistant green sectors in their first true leaves. Thus, normal, low-frequency transmission of paternal plastids in N. tabacum can be directly revealed by using tentoxin.

Challenge of Nicotiana tabacumcv Xanthi with the ethylene‐inducing xylanase (EIX) from Trichoderma viridecauses rapid induction of plant defense responses leading to hypersensitive necrosis. This phenomenon is cultivar‐specific; no response is detected when N. tabacumcv Hicks is similarly treated. The responsiveness is determined in tobacco and tomato by a single dominant gene. EIX was labeled with fluorescein‐isothiocyanate and incubated with cell suspension cultures, protoplasts or microsomal membranes. Binding of EIX to the microsomal membranes was found to be specific and saturable, with a dissociation constant of 6.2 nM. Using confocal laser microscopy, the EIX binding site was localized to the plasma membrane. Binding of EIX to its high‐affinity site occurred in responsive species. These results demonstrate the existence of a high‐affinity binding site for EIX on the plasma membrane of responsive cultivars. Chemical cross‐linking of EIX to microsomal membranes from responding plants revealed a 66 kDa protein complex. This protein may function as the receptor that mediates the hypersensitive response induced by EIX binding.

The receptors for the fungal elicitor EIX (LeEix1 and LeEix2) belong to a class of leucine-rich repeat cell-surface glycoproteins with a signal for receptor-mediated endocytosis. Both receptors are able to bind the EIX elicitor while only the LeEix2 receptor mediates defense responses. We show that LeEix1 acts as a decoy receptor and attenuates EIX induced internalization and signaling of the LeEix2 receptor. We demonstrate that BAK1 binds LeEix1 but not LeEix2. In plants where BAK1 was silenced, LeEix1 was no longer able to attenuate plant responses to EIX, indicating that BAK1 is required for this attenuation. We suggest that LeEix1 functions as a decoy receptor for LeEix2, a function which requires the kinase activity of BAK1.

Leucine-rich-repeat receptor protein (LRR-RLPs) and LRR-RLKs have been linked with signaling and defense responses in plants. EIX (ethylene-inducing xylanase) triggers a defense response via the LeEix2 receptor, while bacterial flagellin triggers plant innate immunity via the FLS2 receptor. Endocytosis has been suggested to be crucial for the process in both cases. Recently, we showed that the EIX elicitor triggers internalization of the LeEix2 receptor. Additionally, we demonstrate that plant EHD2 is an important factor in the internalization and regulation of the induction of plant immunity in the case of EIX/LeEix2 but does not appear to be involved in the flg/FLS2 system. Here we show that EHD2 is also involved in the signaling of the Cf4 and Cf9 receptors, causing inhibition of hypersensitive respond (HR) and ethylene biosynthesis upon over-expression of EHD2. Our results suggest that different endocytosis pathways are involved in the induction of plant defense responses.

Endocytosis regulates many important and diverse processes in eukaryotic life. EH domain containing proteins function as regulators of endocytosis through protein-protein interactions. Several interactors of mammalian EHDs were identified, including clathrin machinery components. The four human EHD proteins share high homology at the protein level and possess similar domains, but appear to be involved in different stages of intracellular trafficking. EHD1 regulates recycling through the endocytic recycling compartment (ERC). EHD2 has been found to inhibit internalization in mammalians when overexpressed. We have recently investigated the importance of EH domain containing proteins in plant endocytosis. We were able to show that both of the Arabidopsis EHD homologs, termed AtEHD1 and AtEHD2, play important roles in plant endocytosis. Knock-down of AtEHD1 delayed internalization, and overexpression of AtEHD2 inhibited endocytosis. Thus, the function of plant EHDs is highly homologous to that of mammalian EHDs.

ABSTRACTPlants are constantly exposed to numerous diverse microbes and pests. They lack an adaptive immune system and rely on innate immunity to perceive and ward off potential pathogens. The plant immune system enables plants to overcome invading microorganisms, and can be defined as highly successful in this regard. Nevertheless, specialized pathogens are able to overcome structural barriers, preformed defenses, innate immunity and are a persistent threat to crop and food supplies worldwide. The rapidly growing world population results in massive demands for agricultural products and reliable crop yields. Therefore, the ability to precisely manipulate plant immunity to resist diverse diseases holds significant promise for enhancing crop production.

A xylanase produced by Trichoderma viride induced defense responses including ethylene biosynthesis and necrosis in Nicotiana tabacum cv. Xanthi leaves. Ethylene pretreatment accentuated the level of ethylene and necrosis produced in response to xylanase. The sensitivity of leaves to xylanase and ethylene was influenced by tissue age. Young leaves were relatively insensitive to both ethylene and xylanase. Mature leaves were relatively insensitive to xylanase but became very sensitive to xylanase after treatment with ethylene. Senescing leaves were more sensitive to xylanase than young or mature leaves. Ethylene alone had little if any effect on ethylene biosynthesis or ACC synthase transcript levels in the absence of xylanase treatment but stimulated accumulation of pathogenesis related protein PR-1 basic transcripts directly. Ethylene-pretreated leaves subsequently treated with xylanase had greatly elevated ACC synthase transcript levels compared to levels in xylanase treated air-pretreated leaves. The time course of PR-1 basic transcript accumulation in response to ethylene was similar to the time course for ethylene enhanced-sensitivity to xylanase as expressed by ethylene biosynthesis, ACC synthase transcript level and necrosis. All the measured effects of ethylene were lost within a similar time frame of 48 h after removing plants from the ethylene atmosphere. A second ethylene treatment of tobacco plants, following the loss of the initial ethylene treatment, restored the enhanced sensitivity of the tissues to xylanase. The continual presence of ethylene is required for maintenance of the effects of ethylene studied here and, the timing of the induction and subsequent loss of the ethylene effects are closely coordinated whether at the molecular or whole tissue level.