' Liberibacter asiaticus' (Las) is a gram-negative bacterial pathogen associated with citrus huanglongbing (HLB) or greening disease. Las is transmitted by the Asian citrus psyllid (ACP) where it colonizes the phloem tissue, resulting in substantial economic losses to citrus industry worldwide. Despite extensive efforts, effective management strategies against HLB remain elusive, necessitating a deeper understanding of the pathogen's biology. Las undergoes cell-to-cell movement through phloem flow and colonizes different tissues in which Las may have varying interactions with the host. Here, we investigate the transcriptomic landscape of Las in citrus seed coat vasculatures, enabling a complete gene expression profiling of Las genome and revealing unique transcriptomic patterns compared to previous studies using midrib tissues. Comparative transcriptomics between seed coat, midrib and ACP identified specific responses and metabolic states of Las in different host tissue. Two Las virulence factors that exhibit higher expression in seed coat can suppress callose deposition. Therefore, they may contribute to unclogging sieve plate pores during Las colonization in seed coat vasculature. Furthermore, analysis of regulatory elements uncovers a potential role of LuxR-type transcription factors in regulating Liberibacter effector gene expression during plant colonization. Together, this work provides novel insights into the pathogenesis of the devastating citrus HLB.

Type 2C protein phosphatases (PP2Cs) are essential for regulating plant immune responses to pathogens. Our study focuses on the tomato PP2C-immunity associated candidate 6 (Pic6), elucidating its role in negatively regulating pattern-triggered immunity (PTI) signaling pathways in tomato. Using reverse transcription quantitative polymerase chain reaction (RT-qPCR), we observed that treatment with microbe-associated molecular patterns (MAMPs)- flg22 and flgII-28-significantly increased mRNA levels in wild-type (RG-PtoR) tomato plants. Pic6 features a conserved N-terminal kinase-interacting motif (KIM) and a C-terminal PP2C domain. We produced variants of Pic6 with mutations in these regions, demonstrating their involvements in negatively regulating tomato immunity. -mediated transient overexpression of Pic6 resulted in enhanced growth of the bacterial pathogen pv. tomato () strain DC3000Δ compared to a YFP control. Additionally, Pic6 overexpression inhibited mitogen-activated protein kinase (MAPK) activation in response to flg22 and flgII-28 treatments. Importantly, Pic6 exhibited phosphatase activity and interacted with tomato Mkk1/Mkk2 proteins and dephosphorylated them in a KIM-dependent manner. Furthermore, we generated RG-pic6 loss-of-function mutants by CRISPR/Cas9, revealing that the absence of Pic6 heightened MAPK activity and increased resistance to strain 85-10 ( 85-10) when compared with the wild-type (RG-PtoR) plants. Transcript analyses showed that after flg22/flgII-28 treatment, PTI-reporter genes and were significantly upregulated in RG-pic6 mutants in comparison to the wild-type (RG-PtoR) plants. Overall, our findings indicate that Pic6 acts as a negative regulator of MAPK signaling and playing a pivotal role in modulating tomato immunity against bacterial pathogens.

Photosynthesis, the remarkable process by which green plants synthesize nutrients using light energy, plays a crucial role in sustaining life on Earth. However, the effects of pathogens on photosynthesis are not widely understood. In general, a reduction of photosynthesis occurs upon the infection with pathogens. Two main scenarios are responsible for the reduction in photosynthetic capacity. In the first scenario, the pathogen attacks green aerial tissues such as caused by fungal and bacterial leaf spots and blights which affect photosynthesis by destroying green leaf tissue or causing defoliation. This leads to a decrease in the photosynthetic area, ultimately reducing photosynthesis. Interestingly, even when the overall chlorophyll content of leaves is significantly reduced due to pathogen invasion, the remaining chlorophyll-containing leaf area may maintain or even enhance its photosynthetic efficiency. This compensatory mechanism helps mitigate the loss of photosynthetic area. However, the overall yield of the plant is still affected. The second scenario is a reduction in chlorophyll content due to chlorosis, which is characterized by yellowing of leaves. It is a common symptom of plant diseases. It refers to a reduction in the amount of chlorophyll per chloroplast, rather than a decrease in chloroplast number. Diseases caused by viruses and phytoplasmas often exhibit chlorosis. While pathogens disrupt photosynthesis, plants exhibit significant adaptations to cope with these challenges. Understanding these interactions is essential for sustainable agriculture and ecosystem health. Thus, in this review, we discuss the effect of several pathogens on the photosynthesis processes and efficiency in detail.

is a highly damaging pathogen that causes high wheat yield losses in temperate climates. emerged during the domestication of wheat in the Fertile Crescent and has been extensively used as a model system for population genetic and genomic studies. New genetic tools and resources have provided a better understanding of the molecular components involved in the wheat- interaction, highlighted by the cloning of three wheat resistance genes and four avirulence genes. Despite the considerable progress made in the last few years, the mechanisms that mediate colonization remain largely unknown. In this review, we summarize the latest advances in understanding the molecular components mediating wheat- interactions and we discuss future research lines to close current knowledge gaps.

Synergistic interactions among unrelated viruses in mixed infections can cause significant yield losses, and viral determinants of these interactions are poorly understood. Wheat ( L.) co-infection with wheat curl mite-transmitted wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) results in disease synergism with a drastically increased symptom phenotype of stunted growth, leaf bleaching, and enhanced titers of both viruses. In this study, we examined the viral determinants responsible for WSMV-TriMV disease synergism through transient expression of select cistrons of WSMV in wheat through TriMV and vice-versa. We found that expression of WSMV P1, NIa, or NIaPro cistrons in wheat through TriMV or vice-versa elicited moderate to severe symptoms with a moderate or no increase in virus titer. However, co-expression of P1 and NIaPro cistrons of WSMV in wheat through TriMV or vice-versa exhibited a WSMV-TriMV disease synergism-like phenotype. Additionally, we found that the P3 cistron of both viruses is dispensable for synergism, whereas HCPro and NIaVPg cistrons of WSMV and TriMV are not the primary determinants but might have a minor role in efficient synergism. In co-infected wheat, accumulation of vsiRNAs was increased, similar to viral genomic RNA copies, despite the presence of dual viral RNA silencing suppressors (VRSS), which function through sequestration of vsiRNAs. Our findings revealed that WSMV-TriMV disease synergism is not caused by the suppression of host post-transcriptional gene silencing by dual VRSS in co-infected wheat and the P1 and NIaPro cistrons of both viruses collectively drive synergistic interactions between WSMV and TriMV in wheat.

Long non-coding RNAs (lncRNAs) are emerging as important regulators of various aspects of immune response and plant-pathogen interactions. However, the regulatory function of lncRNAs during plant-nematode interaction remain largely elusive. In this study, we investigated the differential regulation and function of lncRNAs during two different stages of tomato infection by the root-knot nematode . At the early stage of infection, 2218 and 2827 lncRNAs were regulated locally in the -induced galls and systemically in the neighboring root cells, respectively. However, at the later stage of infection, the number of -regulated lncRNAs was dramatically reduced with only 49 lncRNA being identified as differentially expressed. Differentially expressed lncRNAs were predicted to encode peptides with functionally annotated domains, providing insights into the potential roles of these peptides in regulating gene expression, RNA stability and splicing, and protein-protein-interactions. Among the differentially expressed lcRNAs, 55 were found to contain putative binding sites for 56 miRNAs. Overexpressing 5 of these lncRNAs significantly increased tomato resistance to , supporting the functional importance of lncRNAs for establishing tomato- interaction. Functional analysis of the target mimicry of lncRNAs towards miRNAs resulted in the identification of two novel regulatory modules involving miR47 and miR156e-5p and their targeted genes that regulate tomato responses to parasitism. Taken together, our data provide novel insights into the transcriptional and post-transcriptional regulatory functions of lncRNA, and open a new avenue to engineer crop plants with enhanced nematode resistance by leveraging the regulatory potential of lncRNAs.

Soybean cyst nematode (SCN, ) is a serious root parasite of soybean () that induces extensive gene expression changes associated with pleiotropic biological activities in infected cells. However, the impacts of various SCN Hg Types on host transcriptome reprogramming remain largely unknown. Here, we developed and used two recombinant inbred lines (RIL-72 and RIL-137) to profile transcriptome reprogramming in the infection sites during the resistant and susceptible interactions with SCN Hg Type 1.2.5.7 and Type 0. SCN bioassays indicated that RIL-72 was susceptible to Type 1.2.5.7 but resistant to Type 0, whereas RIL-137 was resistant to both types. Comparative analysis of gene expression changes induced by Type 1.2.5.7 in the resistant and susceptible lines revealed distinct transcriptome regulation with a number of similarly and oppositely regulated genes. The expression levels of similarly regulated genes in the susceptible line appeared to be insufficient to mount an effective defense against SCN. The functional importance of oppositely regulated genes was confirmed using virus induced gene silencing and overexpression approaches. Further transcriptome comparisons revealed shared as well as Hg Type- and genotype-specific transcriptome reprogramming. Shared transcriptome responses were mediated through common SCN-responsive genes and conserved immune signaling, whereas genotype-specific responses were derived from genetic variability, metabolic and hormonal differences, and varied regulation of protein phosphorylation and ubiquitination. The conserved defense mechanisms together with genotype-specific responses would enable plants to trigger effective and tailored immune responses to various Hg types and adapt the defense response to their genetic backgrounds.

Tomato spotted wilt virus (TSWV) is an orthotospovirus that infects both plants and insect vectors. Understanding the protein localization and interactions is crucial for unraveling the infection cycle and host-virus interactions. We investigated and compared the localization of TSWV proteins. A change in localization over time was associated with the viral proteins that did not contain signal peptides and transmembrane domains such as N, NSs and NSm, however, this only occurred in the plant cells, not in the insect cells. The localization between plants and insects otherwise was consistent indicating a similar mechanism is utilized by the virus in both types of cells. We also tested the localization of the proteins during an active plant infection using free RFP as a marker to highlight the nucleus and cytoplasm. Voids in the cytoplasm were shown only during infection and N, NSs, NSm and to lesser extent, G and G, were surrounding these areas suggesting it may be a site of replication or morphogenesis. Furthermore, we tested the interactions of viral proteins using both bimolecular fluorescence complementation (BiFC) and membrane-based yeast two-hybrid (MbY2H) assays. These revealed self-interactions of NSm, N, G, G, and NSs. We also identified interactions between different TSWV proteins, indicating their roles and host interactions, such as between NSs and G and N and G which may be necessary during the replication and assembly processes respectively. This research expands our knowledge of TSWV infection and elaborates on the intricate relationships between viral proteins, cellular dynamics, and host responses.

Great interest exists in developing a transgenic trait that controls the economically important soybean () pest, soybean cyst nematode (SCN, ), due to its adaptation to native resistance. Soybean plants expressing the delta-endotoxin, Cry14Ab, were recently demonstrated to control SCN in both growth chamber and field testing. In that communication, ingestion of the Cry14Ab toxin by SCN second stage juveniles (J2s) was demonstrated using fluorescently labeled Cry14Ab in an in vitro assay. Here, we show that consistent with expectations for a Cry toxin, Cry14Ab has a mode of action unique from the native resistance sources Peking and PI 88788. Further, we demonstrate in planta the ingestion and localization of the Cry14Ab toxin in the midgut of nematodes feeding on roots expressing Cry14Ab using immunogold labeling and transmission electron microscopy. We observed immunolocalization of the toxin and resulting intestinal damage primarily in the microvillus-like structure (MvL)-containing region of the midgut intestine but not in nematodes feeding on roots lacking toxin. This demonstrated that Cry14Ab was taken up by the J2 SCN, presumably through the feeding tube within the plant root cell that serves as its feeding site. This suggests that relatively large proteins can be taken up through the feeding tube. Electron microscopy showed that Cry14Ab caused lysis of the midgut MvL membrane and eventual degradation of the MvL and the lysate, forming particulate aggregates. The accumulated electron-dense aggregate in the posterior midgut intestine was not observed in SCN in nonCry14Ab-expressing plants. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

pv. () causes bacterial leaf streak (BLS) of rice. This disease represents a major constraint for rice production, a crop feeding more than half of the world's population. was first described in 1918 in the Philippines and is prevalent in Southeast Asia. Today, BLS is also omnipresent in both East and West Africa where the disease was first reported in the early 1980s. The appearance of in Africa decades after its first report in Asia suggests that the disease could have been introduced from Asia to Africa. Strict conservation of five Transcription Activator Like (TAL) effectors in whole-genome sequences of 10 strains of including 3 from West-Africa and 7 from Asia also support this hypothesis. East Africa, and especially Madagascar, where the disease was first described in 1985 is located at the interface between Asia and Africa, hence representing an interesting region to explore the link between strains from Asia and West-Africa. In this study, we i) reconstructed the genome of an historical strain from herbarium specimen of rice showing symptoms of BLS, sampled in Madagascar in 1931, 50 years before the first description of the disease, and ii) sequenced 9 new modern strains including 5 from Madagascar and East-Africa. The analysis of those new genomes along with previously published ones shed light within the evolutionary and epidemiological history of .

The soilborne Gram-negative phytopathogenic beta-proteobacterium strain OE1-1 produces methyl 3-hydroxymyristate (3-OH MAME) as the quorum sensing (QS) signal by the methyltransferase PhcB and senses the chemical, activating the LysR family transcriptional regulator PhcA, which regulates the QS-dependent genes responsible for QS-dependent phenotypes including virulence. The sensor histidine kinases PhcS and VsrA are reportedly involved in the regulation of QS-dependent genes. To elucidate the function of PhcS and VsrA in the active QS, we generated the deletion and -deletion mutants, which exhibited weak changes to their QS-dependent phenotypes including virulence. The and -deletion mutant (Δ/) had significant changes in its QS-dependent phenotypes and was nonvirulent, similar to the -deletion mutant. The mutant (PhcS-H230Q) with a substitution of histidine to glutamine at amino acid position 230 in PhcS but not the mutant (VsrA-H256Q) with a substitution of histidine to glutamine at amino acid position 256 in VsrA exhibited significant changes in QS-dependent phenotypes and lost virulence. The transcriptome analysis with RNA-sequencing revealed significant alterations to the expression of QS-dependent genes in the Δ/ and PhcS-H230Q but not VsrA-H256Q, similar to the -deletion mutant. The exogenous 3-OH MAME application led to a significantly enhanced QS-inducible major exopolysaccharide EPS I production of the strain OE1-1 and -deletion mutant but not Δ/ and PhcS-H230Q. Collectively, results of the present genetic study suggested that PhcS contributes to QS along with VsrA and that histidine at amino acid position 230 of PhcS is required for 3-OH MAME sensing, thereby influencing QS-dependent phenotypes including virulence of the strain OE1-1. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

The ability of laser scanning confocal microscopy to generate high-contrast 2D and 3D images has become essential in studying plant-fungal interactions. Techniques such as visualization of native fluorescence, fluorescent protein tagging of microbes, GFP/RFP-fusion proteins, and fluorescent labelling of plant and fungal proteins have been widely used to aid in these investigations. Use of fluorescent proteins has several pitfalls including variability of expression in planta and the requirement of gene transformation. Here we used the unlabeled pathogens , f. , and infecting wheat, barley, and sugar beet respectively, to show the utility of a staining and imaging pipeline that uses propidium iodide (PI), which stains RNA and DNA, and wheat germ agglutinin labeled with fluorescein isothiocyanate (WGA-FITC), which stains chitin, to visualize fungal colonization of plants. This pipeline relies on the use of KOH to remove the cutin layer of the leaf, increasing its permeability, allowing the different stains to penetrate and effectively bind to their targets, resulting in a consistent visualization of cellular structures. To expand the utility of this pipeline, we used the staining techniques in conjunction with machine learning to analyze fungal biomass through volume analysis, as well as quantifying nuclear breakdown, an early indicator of programmed cell death (PCD). This pipeline is simple to use, robust, consistent across host and fungal species and can be applied to most plant-fungal interactions. Therefore, this pipeline can be used to characterize model systems as well as non-model interactions where transformation is not routine.

Septoria tritici blotch is caused by the fungus and poses a major threat to wheat productivity. There are over twenty mapped loci in wheat that confer strong (gene-for-gene) resistance against this pathogen, however the corresponding genes in that confer virulence against distinct genes remain largely unknown. In this study, we developed a rapid forward genetics methodology to identify genes that enable to gain virulence on previously resistant wheat varieties. We used the known gene-for-gene interaction between and as a proof-of-concept that this method could quickly recover single candidate virulence genes. We subjected the avirulent strain IPO323, which carries the recognized allele, to UV mutagenesis and generated a library of over 66,000 mutants. We screened these mutants on leaves of the resistant wheat variety Cadenza, in mixtures (soups) ranging from 100-500 mutants per soup. We identified five soups with a gain-of-virulence (GoV) phenotype relative to the IPO323 parental strain and re-sequenced 18 individual isolates, including four control isolates and two mutants lacking virulence, when screened individually. Of the 12 confirmed GoV mutants, one had a single nucleotide polymorphism (SNP) in the coding region. The other 11 GoV mutants exhibited large (~70Kb) deletions at the end of chromosome 5, including the locus. Our findings demonstrate the efficiency of this forward genetic approach in elucidating the genetic basis of qualitative resistance to and the potential to rapidly identify other, currently unknown, genes in this pathogen.

After having co-existed in plant genomes for at least 200 million years, the products of microRNA (miRNA) and Nucleotide-Binding Leucine Rich Repeat protein (NLR) genes formed a regulatory relationship in the common ancestor of modern gymnosperms and angiosperms. From then on, DNA polymorphisms occurring at miRNA target sequences within NLR transcripts must have been compensated by mutations in the corresponding mature miRNA sequence. The potential evolutionary advantage of such regulation remains largely unknown and might be related to two non-exclusive scenarios: miRNA-dependent regulation of NLR levels might prevent defense mis-activation with negative effects on plant growth and reproduction; or reduction of active miRNA levels in response to pathogen derived molecules (PAMPS and silencing suppressors) might rapidly release otherwise silent NLR transcripts for rapid translation and thereby enhance defense. Here, we used plants deficient for miR472 function to study the impact of releasing its NLR targets on plant growth and reproduction and on defense against the fungal pathogen . We show that miR472 regulation has a dual role, participating both in the tight regulation of plant defense and growth. MIM472 lines, with reduced active miR472, are more resistant to pathogens and, correlatively, have reduced relative growth compared to wild-type plants although the end of their reproductive phase is delayed, exhibiting higher adult biomass and similar seed yield as the wild-type. Our study highlights how negative consequences of defense activation might be compensated by changes in phenology and that miR472 reduction is an integral part of plant defense responses.

infects a wide variety of crops. The () is conserved across many strains and is responsible for producing an extracellular chemical signal, leudiazen. Disruption of the gene in pv. () UMAF0158 alleviated tomato chlorosis caused by this bacterium. We showed that deletion of entire reduced UMAF0158 population in tomato leaflets. Leudiazen restored the signaling activity of the deletion mutant at a concentration as low as 10 nM. Both the diazeniumdiolate and isobutyl groups of leudiazen are critical for this potent signaling activity. Transcriptional analysis showed that and leudiazen induce the expression of as well as an uncharacterized gene cluster, RS17235-RS17245. We found that this cluster enhances survival of UMAF0158 in planta and is widely distributed in strains. Our results demonstrate that plays prominent roles in the virulence and growth of . The and -like signaling systems in different bacteria likely regulate diverse microbe-host interactions.

The soybean cyst nematode (SCN; ) facilitates infection by secreting a repertoire of effector proteins into host cells to establish a permanent feeding site composed of a syncytium of root cells. Among the diverse proteins secreted by the nematode, we were specifically interested in identifying proteases to pursue our goal of engineering decoy substrates that elicit an immune response when cleaved by an SCN protease. We identified a cysteine protease that we named Cysteine Protease 1 (CPR1), which was predicted to be a secreted effector based on transcriptomic data obtained from SCN esophageal gland cells, presence of a signal peptide, and lack of transmembrane domains. CPR1 is conserved in all isolates of SCN sequenced to date, suggesting it is critical for virulence. Transient expression of CPR1 in leaves suppressed cell death induced by a constitutively active nucleotide binding leucine-rich repeat protein, RPS5, indicating that CPR1 inhibits effector-triggered immunity. CPR1 localizes in part to the mitochondria when expressed in planta. Proximity-based labeling in transgenic soybean roots, co-immunoprecipitation, and cleavage assays identified a branched-chain amino acid aminotransferase from soybean (GmBCAT1) as a substrate of CPR1. Consistent with this, GmBCAT1 also localizes to mitochondria. Silencing of the transcript in the nematode reduced penetration frequency in soybean roots while the expression of in soybean roots enhanced susceptibility. Our data demonstrates that CPR1 is a conserved effector protease with a direct target in soybean roots, highlighting it as a promising candidate for decoy engineering.

Molecular inter-species dialogue between leguminous plants and nitrogen-fixing rhizobia results in the development of symbiotic root nodules. This is initiated by several nodulation-related receptors present on the surface of root hair epidermal cells. We have shown previously that specific subunits of heterotrimeric G proteins and their regulatory RGS (regulator of G-protein signaling) proteins act as molecular links between the receptors and downstream components during nodule formation in soybeans. Nod factor receptor 1 (NFR1) interacts with and phosphorylates RGS proteins to regulate the G-protein cycle. Symbiosis receptor-like kinases (SymRK) phosphorylate Gα to make it inactive and unavailable for Gβγ. We now show that like NFR1, SymRK also interacts with the RGS proteins to phosphorylate them. Phosphorylated RGS has higher GTP accelerating activity, which favors conversion of active Gα to its inactive form. Phosphorylation of RGS proteins is physiologically relevant, as overexpression of a phospho-mimic version of RGS protein enhances nodule formation in soybean. These results reveal an intricate fine-tuning of the G-protein signaling during nodulation, where a negative regulator (Gα) is effectively deactivated by RGS due to the concerted efforts of several receptor proteins to ensure adequate nodulation.

A path to sustainably reduce world hunger, food insecurity, and malnutrition is to close the crop yield gap, particularly, losses due to pathogens. Breeding resistant crops is key to achieving this goal, an effort requiring collaboration among stakeholders, scientists, breeders, farmers and policymakers. During a disease outbreak, epidemiologists survey the occurrence of a disease after which pathologists investigate mechanisms to stop an infection. Policymakers then implement strategies with farmers and breeders to overcome the outbreak. Information flow from the field to the lab and back to the field involves several processing hubs that require different information inputs. Failure to communicate the necessary information results in the transfer of meaningless data. Here, we discuss gaps in information acquisition and transfer between the field and laboratory. Using rice bacterial blight disease as an example, we discuss pathogen biology and disease resistance to point out the importance of reporting pathogen strains that caused an outbreak to optimize the deployment of resistant crop varieties. We examine differences between infection in the field and assays performed in the laboratory to draw awareness of possible misinformation concerning plant resistance or susceptibility. We discuss key data considered useful for reporting disease outbreaks, sampling bias, and suggestions for improving data quality. We also touch on the knowledge gap in the state-of-the-art literature regarding disease dispersal and transmission. We use a recent case study to exemplify the gaps mentioned. We conclude by highlighting potential actions that may contribute to food security and to closing of the yield gap.