Blight-resistant rice lines are the most effective solution for bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo). Key resistance mechanisms involve SWEET genes as susceptibility ...factors. Bacterial transcription activator-like (TAL) effectors bind to effector-binding elements (EBEs) in SWEET gene promoters and induce SWEET genes. EBE variants that cannot be recognized by TAL effectors abrogate induction, causing resistance. Here we describe a diagnostic kit to enable analysis of bacterial blight in the field and identification of suitable resistant lines. Specifically, we include a SWEET promoter database, RT-PCR primers for detecting SWEET induction, engineered reporter rice lines to visualize SWEET protein accumulation and knock-out rice lines to identify virulence mechanisms in bacterial isolates. We also developed CRISPR-Cas9 genome-edited Kitaake rice to evaluate the efficacy of EBE mutations in resistance, software to predict the optimal resistance gene set for a specific geographic region, and two resistant 'mega' rice lines that will empower farmers to plant lines that are most likely to resist rice blight.
Bacterial blight of rice is an important disease in Asia and Africa. The pathogen, Xanthomonas oryzae pv. oryzae (Xoo), secretes one or more of six known transcription-activator-like effectors ...(TALes) that bind specific promoter sequences and induce, at minimum, one of the three host sucrose transporter genes SWEET11, SWEET13 and SWEET14, the expression of which is required for disease susceptibility. We used CRISPR-Cas9-mediated genome editing to introduce mutations in all three SWEET gene promoters. Editing was further informed by sequence analyses of TALe genes in 63 Xoo strains, which revealed multiple TALe variants for SWEET13 alleles. Mutations were also created in SWEET14, which is also targeted by two TALes from an African Xoo lineage. A total of five promoter mutations were simultaneously introduced into the rice line Kitaake and the elite mega varieties IR64 and Ciherang-Sub1. Paddy trials showed that genome-edited SWEET promoters endow rice lines with robust, broad-spectrum resistance.
SignificancePlants evolved in an environment colonized by a vast number of microbes, which collectively constitute the plant microbiota. The majority of microbiota taxa are nonpathogenic and may be ...beneficial to plants under certain ecological or environmental conditions. We conducted experiments to understand the features of long-term interactions of nonpathogenic microbiota members with plants. We found that a multiplication-death equilibrium explained the shared long-term static populations of nonpathogenic bacteria and that in planta bacterial transcriptomic signatures were characteristic of the stationary phase, a physiological state in which stress protection responses are induced. These results may have significant implications in understanding the bulk of "nonpathogenic" plant-microbiota interactions that occur in agricultural and natural ecosystems.
Summary
Transcription Activator‐Like effectors (TALes) represent the largest family of type III effectors among pathogenic bacteria and play a critical role in the process of infection. Strains of ...Xanthomonas oryzae pv. oryzae (Xoo) and some strains of other Xanthomonas pathogens contain large numbers of TALe genes. Previous techniques to clone individual or a complement of TALe genes through conventional strategies are inefficient and time‐consuming due to multiple genes (up to 29 copies) in a given genome, and technically challenging due to the repetitive sequences (up to 33 nearly identical 102‐nucleotide repeats) of individual TALe genes. Thus, only a limited number of TALe genes have been molecularly cloned and characterized, and the functions of most TALe genes remain unknown. Here, we present an easy and efficient cloning technique to clone TALe genes selectively through in vitro homologous recombination and single‐strand annealing, and demonstrate the feasibility of this approach with four different Xoo strains. Based on the Gibson assembly strategy, two complementary vectors with scaffolds that can preferentially capture all TALe genes from a pool of genomic fragments were designed. Both vector systems enabled cloning of a full complement of TALe genes from each of four Xoo strains and functional analysis of individual TALes in rice in approximately 1 month compared to 3 months by previously used methods. The results demonstrate a robust tool to advance TALe biology and a potential for broad usage of this approach to clone multiple copies of highly competitive DNA elements in any genome of interest.
Some fungal epithiodiketopiperazine alkaloids display α,β‐polysulfide bridges alongside diverse structural variations. However, the logic of their chemical diversity has rarely been explored. Here, ...we report the identification of three new (2, 3, 8) and five known (1, 4–7) epithiodiketopiperazines of this subtype from a marine‐derived Penicillium sp. The structure elucidation was supported by multiple spectroscopic analyses. Importantly, we observed multiple nonenzymatic interconversions of these analogues in aqueous solutions and organic solvents. Furthermore, the same biosynthetic origin of these compounds was supported by one mined gene cluster. The dominant analogue (1) demonstrated selective cytotoxicity to androgen‐sensitive prostate cancer cells and HIF‐depleted colorectal cells and mild antiaging activities, linking the bioactivity to oxidative stress. These results provide crucial insight into the formation of fungal epithiodiketopiperazines through chemical interconversions.
Chemical and functional diversity: Multiple new and known cytotoxic epithiodiketopiperazines containing α,β‐polysulfide bridges were produced by a Penicillium sp. isolated from a black band layer of a coral sample. These analogues originated from the same gene cluster but demonstrated nonenzymatic interconversions to afford chemical diversity.
Waikikiamides A-C (
-
), structurally complex diketopiperazine derivatives, and putative biogenic precursors, (+)-semivioxanthin (
), notoamide F (
), and (-)-notoamide A (
), were isolated from
sp. ...FM242.
and
, bearing a hendecacyclic ring system, represent a novel skeleton.
features the first unique heterodimer of two notoamide analogs with an N-O-C bridge. Compounds
and
exhibit antiproliferative activity with IC
values in the range of 0.56 to 1.86 μM. The gene clusters mined from the sequenced genome support their putative biosynthetic pathways.
Parasites and other symbionts are crucial components of ecosystems, regulating host populations and supporting food webs. However, most symbiont systems, especially those involving commensals and ...mutualists, are relatively poorly understood. In this study, we have investigated the nature of the symbiotic relationship between birds and their most abundant and diverse ectosymbionts: the vane‐dwelling feather mites. For this purpose, we studied the diet of feather mites using two complementary methods. First, we used light microscopy to examine the gut contents of 1,300 individual feather mites representing 100 mite genera (18 families) from 190 bird species belonging to 72 families and 19 orders. Second, we used high‐throughput sequencing (HTS) and DNA metabarcoding to determine gut contents from 1,833 individual mites of 18 species inhabiting 18 bird species. Results showed fungi and potentially bacteria as the main food resources for feather mites (apart from potential bird uropygial gland oil). Diatoms and plant matter appeared as rare food resources for feather mites. Importantly, we did not find any evidence of feather mites feeding upon bird resources (e.g., blood, skin) other than potentially uropygial gland oil. In addition, we found a high prevalence of both keratinophilic and pathogenic fungal taxa in the feather mite species examined. Altogether, our results shed light on the long‐standing question of the nature of the relationship between birds and their vane‐dwelling feather mites, supporting previous evidence for a commensalistic–mutualistic role of feather mites, which are revealed as likely fungivore–microbivore–detritivore symbionts of bird feathers.
Plant-pathogenic Streptomyces spp. cause scab disease on economically important root and tuber crops, the most important of which is potato. Key virulence determinants produced by these species ...include the cellulose synthesis inhibitor, thaxtomin A, and the secreted Nec1 protein that is required for colonization of the plant host. Recently, the genome sequence of Streptomyces scabies 87-22 was completed, and a biosynthetic cluster was identified that is predicted to synthesize a novel compound similar to coronafacic acid (CFA), a component of the virulence-associated coronatine phytotoxin produced by the plant-pathogenic bacterium Pseudomonas syringae. Southern analysis indicated that the cfa-like cluster in S. scabies 87-22 is likely conserved in other strains of S. scabies but is absent from two other pathogenic streptomycetes, S. turgidiscabies and S. acidiscabies. Transcriptional analyses demonstrated that the cluster is expressed during plant–microbe interactions and that expression requires a transcriptional regulator embedded in the cluster as well as the bldA tRNA. A knockout strain of the biosynthetic cluster displayed a reduced virulence phenotype on tobacco seedlings compared with the wild-type strain. Thus, the cfa-like biosynthetic cluster is a newly discovered locus in S. scabies that contributes to host–pathogen interactions.
While most plant-pathogenic
species cause scab disease on a variety of plant hosts,
is the sole causative agent of soil rot disease of sweet potato and closely related plant species. Here, genome ...sequencing of virulent and avirulent
strains coupled with comparative genomic analyses has identified genome content and organization features unique to this streptomycete plant pathogen. The results here will enable future research into the mechanisms used by
to cause disease and to persist in its niche environment.
is an oomycete plant pathogen with a wide host range. Worldwide,
is known for causing the principal disease of chili pepper crops. Our goal was to expand the available genome resources for this ...diverse pathogen by generating whole-genome sequences for six isolates of
.
from Mexico.
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