Pseudomonas syringae Type III Effector HopZ1 Targets a Host Enzyme to Suppress Isoflavone Biosynthesis and Promote Infection in Soybean

SummaryType III secreted effectors (T3SEs), such as Pseudomonas syringae HopZ1, are essential bacterial virulence proteins injected into the host cytosol to facilitate infection. However, few direct targets of T3SEs are known. Investigating the target(s) of HopZ1 in soybean, a natural P. syringae host, we find that HopZ1 physically interacts with the isoflavone biosynthesis enzyme, 2-hydroxyisoflavanone dehydratase (GmHID1). P. syringae infection induces gmhid1 expression and production of daidzein, a major soybean isoflavone. Silencing gmhid1 increases susceptibility to P. syringae infection, supporting a role for GmHID1 in innate immunity. P. syringae expressing active but not the catalytic mutant of HopZ1 inhibits daidzein induction and promotes bacterial multiplication in soybean. HopZ1-enhanced P. syringae multiplication is at least partially dependent on GmHID1. Thus, GmHID1 is a virulence target of HopZ1 to promote P. syringae infection of soybean. This work highlights the isoflavonoid biosynthesis pathway as an antibacterial defense mechanism and a direct T3SE target

Non-TAL Effectors From Xanthomonas oryzae pv. oryzae Suppress Peptidoglycan-Triggered MAPK Activation in Rice

Xanthomonas oryzae pv. oryzae, the causal pathogen of bacterial blight of rice, depends on its type III secretion system and associated effector proteins to grow and colonize the vascular tissues of rice plants. The type III effectors include a family of closely related transcription activator-like (TAL) effectors and the rest of diverse effectors, so-called non-TAL effectors. Our understanding of non-TAL effectors for pathogenesis in rice blight is still limited. Here we report a feasible method to rapidly detect the activation of mitogen-activated protein kinase pathway in rice mesophyll protoplasts by the X. oryzae pv. oryzae derived peptidoglycan and screen for virulent effectors that can suppress the pathogen-associated molecular pattern triggered immunity (PTI) response. Amongst 17 non-TAL effectors transiently expressed in rice cells, we found that three effectors (XopZ, XopN, and XopV) were able to suppress the peptidoglycan-triggered MAPK activation. The triple mutant of the X. oryzae pv. oryzae strain PXO99A lacking XopZ, XopN, and XopV showed additively reduced virulence. Adding back either of genes restored the virulence of the triple mutant. Our results demonstrate the collective and redundant ability of defense suppression by non-TAL effectors in causing bacterial blight of rice

Base-Editing-Mediated Artificial Evolution of OsALS1 In Planta to Develop Novel Herbicide-Tolerant Rice Germplasms

Recently developed CRISPR-mediated base editors, which enable the generation of numerous nucleotide changes in target genomic regions, have been widely adopted for gene correction and generation of crop germplasms containing important gain-of-function genetic variations. However, to engineer target genes with unknown functional SNPs remains challenging. To address this issue, we present here a base-editing-mediated gene evolution (BEMGE) method, employing both Cas9n-based cytosine and adenine base editors as well as a single-guide RNA (sgRNA) library tiling the full-length coding region, for developing novel rice germplasms with mutations in any endogenous gene. To this end, OsALS1 was artificially evolved in rice cells using BEMGE through both Agrobacterium-mediated and particle-bombardment-mediated transformation. Four different types of amino acid substitutions in the evolved OsALS1, derived from two sites that have never been targeted by natural or human selection during rice domestication, were identified, conferring varying levels of tolerance to the herbicide bispyribac-sodium. Furthermore, the P171F substitution identified in a strong OsALS1 allele was quickly introduced into the commercial rice cultivar Nangeng 46 through precise base editing with the corresponding base editor and sgRNA. Collectively, these data indicate great potential of BEMGE in creating important genetic variants of target genes for crop improvement.publishedVersio

Soft Sensors for Pulp Freeness and Outlet Consistency Estimation in the Alkaline Peroxide Mechanical Pulping (APMP) High-Consistency Refining Process

In the mechanical pulping process, some process state and product quality variables are difficult to measure on-line. In this paper, soft sensors were used to estimate Canadian Standard Freeness (CSF) and outlet consistency (Cout) after the high consistency refining stage of the alkaline peroxide mechanical pulping (APMP) process. After the secondary variables for modeling that are readily available processed measurements in pre-treatment and the HC refining stage was selected, models based on the case-based reasoning (CBR) method were developed to estimate CSF and Cout. The ability of CBR soft sensors to predict CSF and Cout was tested using data collected from an APMP mill, and the results were satisfactory. Additionally, two typical soft sensor methods that back propagation network (BP) algorithms and support vector regression algorithms (SVR) were employed to predict CSF and Cout and evaluate the performance of the CBR soft sensor. As a result, the proposed soft sensor demonstrated a better performance than the BP method and can be regarded as of comparable quality to the SVR method

Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice

The type II CRISPR/Cas system from Streptococcus pyogenes and its simplified derivative, the Cas9/ single guide RNA (sgRNA) system, have emerged as potent new tools for targeted gene knockout in bacteria, yeast, fruit fly, zebrafish and human cells. Here, we describe adaptations of these systems leading to successful expression of the Cas9/ sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum. Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco. The mutant GFP gene contained target sites in its 50 coding regions that were successfully cleaved by a CAS9/sgRNA complex that, along with error-prone DNA repair, resulted in creation of functional GFP genes. DNA sequencing confirmed Cas9/sgRNA-mediated mutagenesis at the target site. Rice protoplast cells transformed with Cas9/sgRNA constructs targeting the promoter region of the bacterial blight susceptibility genes, OsSWEET14 and OsSWEET11, were confirmed by DNA sequencing to contain mutated DNA sequences at the target sites. Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its nearterm use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications. Download is lower-res 2M file. High-res 18M file and supplementary data are attached below

SpRY greatly expands the genome editing scope in rice with highly flexible PAM recognition

Background Plant genome engineering mediated by various CRISPR-based tools requires specific protospacer adjacent motifs (PAMs), such as the well-performed NGG, NG, and NNG, to initiate target recognition, which notably restricts the editable range of the plant genome. Results In this study, we thoroughly investigate the nuclease activity and the PAM preference of two structurally engineered SpCas9 variants, SpG and SpRY, in transgenic rice. Our study shows that SpG nuclease favors NGD PAMs, albeit less efficiently than the previously described SpCas9-NG, and that SpRY nuclease achieves efficient editing across a wide range of genomic loci, exhibiting a preference of NGD as well as NAN PAMs. Furthermore, SpRY-fused cytidine deaminase hAID*Δ and adenosine deaminase TadA8e are generated, respectively. These constructs efficiently induce C-to-T and A-to-G conversions in the target genes toward various non-canonical PAMs, including non-G PAMs. Remarkably, high-frequency self-editing events (indels and DNA fragments deletion) in the integrated T-DNA fragments as a result of the nuclease activity of SpRY are observed, whereas the self-editing of SpRY nickase-mediated base editor is quite low in transgenic rice lines. Conclusions The broad PAM compatibility of SpRY greatly expands the targeting scope of CRISPR-based tools in plant genome engineering.publishedVersio

Allelic variants of the Pseudomonas syringae type III effector HopZ1 are differentially recognized by plant resistance systems

The bacterial plant pathogen Pseudomonas syringae depends on the type III secretion system and type III-secreted effectors to cause disease in plants. HopZ is a diverse family of type III effectors widely distributed in P. syringae isolates. Among the HopZ homologs, HopZ1 is ancient to P. syringae and has been shown to be under strong positive selection driven by plant resistance-imposed selective pressure. Here, we characterized the virulence and avirulence functions of the three HopZ1 alleles in soybean and Nicotiana benthamiana. In soybean, HopZ1 alleles have distinct functions: HopZ1a triggers defense response, HopZ1b promotes bacterial growth, and HopZ1c has no observable effect. In N. benthamiana, HopZ1a and HopZ1b both induce plant defense responses. However, they appear to trigger different resistance pathways, evidenced by two major differences between HopZ1a- and HopZ1b-triggered hypersensitive response (HR): i) the putative N-acylation sites had no effect on HopZ1a-triggered cell death, whereas it greatly enhanced HopZ1b-triggered cell death; and ii) the HopZ1b-triggered HR, but not the HopZ1a-triggered HR, was suppressed by another HopZ homolog, HopZ3. We previously demonstrated that HopZ1a most resembled the ancestral allelic form of HopZ1; therefore, this new evidence suggested that differentiated resistance systems have evolved in plant hosts to adapt to HopZ1 diversification in P. syringae

A New Era in Herbicide-Tolerant Crops Development by Targeted Genome Editing

Weeds are one of the biggest problems that modern agriculture is facing worldwide due to the impact they have on crop productivity. Thus, there is a necessity to develop crop varieties with herbicide resistance or tolerance, which would provide cost-effective tools for helping farmers control weeds in the field. Development of herbicide-tolerant crops was initially based on conventional plant breeding and transgenic technology. In recent years, the emerging genome technologies, including ZFNs (zinc-finger nucleases), TALENs (transcription activator-like effector nucleases), and CRISPR (clustered regularly interspaced short palindromic repeat), provide us a new way for crop improvement through precise manipulation of endogenous genes in the plant genomes. Among these, CRISPR technologies, including nuclease systems, base editors, and prime editors, are really promising in creating novel crop germplasms with herbicide tolerance as they are simple, easy to use, and highly efficient. In this review, we briefly summarize the latest development and breakthroughs of CRISPR technologies in creating herbicide-tolerant crops. Finally, we discuss the future applications of CRISPR technologies in developing herbicide-tolerant crops.acceptedVersio