Agrobacterium-mediated plant transformation (AMT) is the basis of modern-day plant biotechnology. One major drawback of this technology is the recalcitrance of many plant species/varieties to Agrobacterium infection, most likely caused by elicitation of plant defense responses. Here, we develop a strategy to increase AMT by engineering Agrobacterium tumefaciens to express a type III secretion system (T3SS) from Pseudomonas syringae and individually deliver the P. syringae effectors AvrPto, AvrPtoB, or HopAO1 to suppress host defense responses. Using the engineered Agrobacterium, we demonstrate increase in AMT of wheat, alfalfa and switchgrass by ~250%–400%. We also show that engineered A. tumefaciens expressing a T3SS can deliver a plant protein, histone H2A-1, to enhance AMT. This strategy is of great significance to both basic research and agricultural biotechnology for transient and stable transformation of recalcitrant plant species/varieties and to deliver proteins into plant cells in a non-transgenic manner.

Anaplasma phagocytophilum is an obligate intracellular Gram-negative bacterium that causes human granulocytic anaplasmosis. Assessing the number of these bacteria is important for in vitro and in vivo infection studies. Colony count is used to set references for the multiplicity of infections in the case of culturable bacteria. However, the number of bacteria present inside the host cells, in which the bacteria are maintained, can be considered in the case of obligate intracellular bacteria. McFarland standards are a series of turbidity-based standards used to visually assess the approximate number of culturable bacteria. The turbidity of each standard can be related to their respective absorbances or optical densities (ODs). In this study, we describe a simple method to assess the approximate number of A. phagocytophilum based on McFarland standards. The ODs of cell-free crude extracts of A. phagocytophilum were used to assess the approximate number of bacteria while considering that the cell debris also contributes to the ODs. The consistency of this method was also tested using the bacterial cultures grown at different times. In summary, we provide a simple method to estimate the number of obligate intracellular bacteria for use in in vitro infection studies.

Summary: Alfalfa (Medicago sativa L.) is one of the most important forage crops throughout the world. Maximizing leaf retention during the haymaking process is critical for achieving superior hay quality and maintaining biomass yield. Leaf abscission process affects leaf retention. Previous studies have largely focused on the molecular mechanisms of floral organ, pedicel and seed abscission but scarcely touched on leaf and petiole abscission. This study focuses on leaf and petiole abscission in the model legume Medicago truncatula and its closely related commercial species alfalfa. By analysing the petiolule‐like pulvinus (plp) mutant in M. truncatula at phenotypic level (breakstrength and shaking assays), microscopic level (scanning electron microscopy and cross‐sectional analyses) and molecular level (expression level and expression pattern analyses), we discovered that the loss of function of PLP leads to an absence of abscission zone (AZ) formation and PLP plays an important role in leaflet and petiole AZ differentiation. Microarray analysis indicated that PLP affects abscission process through modulating genes involved in hormonal homeostasis, cell wall remodelling and degradation. Detailed analyses led us to propose a functional model of PLP in regulating leaflet and petiole abscission. Furthermore, we cloned the PLP gene (MsPLP) from alfalfa and produced RNAi transgenic alfalfa plants to down‐regulate the endogenous MsPLP. Down‐regulation of MsPLP results in altered pulvinus structure with increased leaflet breakstrength, thus offering a new approach to decrease leaf loss during alfalfa haymaking process.

Https://doi.org/10.18632/oncotarget.27569 Salum J. Lidenge, For Yue Tso, Owen Ngalamika, Jaydeep Kolape, John R. Ngowi, Julius Mwaiselage, Charles Wood, John T. West

Serendipita vermifera (=Sebacina vermifera; isolate MAFF305830) is a mycorrhizal fungus originally isolated from the roots of an Australian orchid that we have previously shown to be beneficial in enhancing biomass yield and drought tolerance in switchgrass, an important bioenergy crop for cellulosic ethanol production in the United States. However, almost nothing is known about how this root-associated fungus proliferates and grows through the soil matrix. Such information is critical to evaluate the possibility of non-target effects, such as unintended spread to weedy plants growing near a colonized switchgrass plant in a field environment. A microcosm experiment was conducted to study movement of vegetative mycelia of S. vermifera between intentionally inoculated switchgrass (Panicum virgatum L.) and nearby weeds. We constructed size-exclusion microcosms to test three different common weeds, large crabgrass (Digitaria sanguinalis L.), Texas panicum (Panicum texanum L.), and Broadleaf signalgrass (Brachiaria platyphylla L.), all species that typically co-occur in Southern Oklahoma and potentially compete with switchgrass. We report that such colonization of non-target plants by S. vermifera can indeed occur, seemingly via co-mingled root systems. As a consequence of colonization, significant enhancement of growth was noted in signalgrass, while a mild increase (albeit not significant) was evident in crabgrass. Migration of the fungus seems unlikely in root-free bulk soil, as we failed to see transmission when the roots were kept separate. This research is the first documentation of non-targeted colonization of this unique root symbiotic fungus and highlights the need for such assessments prior to deployment of biological organisms in the field.

When positioned horizontally, roots grow down toward the direction of gravity. This phenomenon, called gravitropism, is influenced by most of the major plant hormones including brassinosteroids. Epi-brassinolide (eBL) was previously shown to enhance root gravitropism, a phenomenon similar to the response of roots exposed to the actin inhibitor, latrunculin B (LatB). This led us to hypothesize that eBL might enhance root gravitropism through its effects on filamentous-actin (F-actin). This hypothesis was tested by comparing gravitropic responses of maize (Zea mays) roots treated with eBL or LatB. LatB- and eBL-treated roots displayed similar enhanced downward growth compared with controls when vertical roots were oriented horizontally. Moreover, the effects of the two compounds on root growth directionality were more striking on a slowly-rotating two-dimensional clinostat. Both compounds inhibited autotropism, a process in which the root straightened after the initial gravistimulus was withdrawn by clinorotation. Although eBL reduced F-actin density in chemically-fixed Z. mays roots, the impact was not as strong as that of LatB. Modification of F-actin organization after treatment with both compounds was also observed in living roots of barrel medic (Medicago truncatula) seedlings expressing genetically encoded F-actin reporters. Like in fixed Z. mays roots, eBL effects on F-actin in living M. truncatula roots were modest compared with those of LatB. Furthermore, live cell imaging revealed a decrease in global F-actin dynamics in hypocotyls of etiolated M. truncatula seedlings treated with eBL compared to controls. Collectively, our data indicate that eBL-and LatB-induced enhancement of root gravitropism can be explained by inhibited autotropic root straightening, and that eBL affects this process, in part, by modifying F-actin organization and dynamics.