Open Access and Reproducibility in Plant Pathology Research: Guidelines and Best Practices.

The landscape of scientific publishing is experiencing a transformative shift toward open access, a paradigm that mandates the availability of research outputs such as data, code, materials, and publications. Open access provides increased reproducibility and allows for reuse of these resources. This article provides guidance for best publishing practices of scientific research, data, and associated resources, including code, in The American Phytopathological Society journals. Key areas such as diagnostic assays, experimental design, data sharing, and code deposition are explored in detail. This guidance aligns with that observed by other leading journals. We hope the information assembled in this paper will raise awareness of best practices and enable greater appraisal of the true effects of biological phenomena in plant pathology.

Recent developments in plant-downy mildew interactions.

Downy mildews are obligate oomycete pathogens that attack a wide range of plants and can cause significant economic impacts on commercial crops and ornamental plants. Traditionally, downy mildew disease control relied on an integrated strategies, that incorporate cultural practices, deployment of resistant cultivars, crop rotation, application of contact and systemic pesticides, and biopesticides. Recent advances in genomics provided data that significantly advanced understanding of downy mildew evolution, taxonomy and classification. In addition, downy mildew genomics also revealed that these obligate oomycetes have reduced numbers of virulence factor genes in comparison to hemibiotrophic and necrotrophic oomycetes. However, downy mildews do deploy significant arrays of virulence proteins, including so-called RXLR proteins that promote virulence or are recognized as avirulence factors. Pathogenomics are being applied to downy mildew population studies to determine the genetic diversity within the downy mildew populations and manage disease by selection of appropriate varieties and management strategies. Genome editing technologies have been used to manipulate host disease susceptibility genes in different plants including grapevine and sweet basil and thereby provide new soucres of resistance genes against downy mildews. Previously, it has proved difficult to transform and manipulate downy mildews because of their obligate lifestyle. However, recent exploitation of RNA interference machinery through Host-Induced Gene Silencing (HIGS) and Spray-Induced Gene Silencing (SIGS) indicate that functional genomics in downy mildews is now possible. Altogether, these breakthrough technologies and attendant fundamental understanding will advance our ability to mitigate downy mildew diseases.

Small RNA-based plant protection against diseases.

Plant diseases cause significant decreases in yield and quality of crops and consequently pose a very substantial threat to food security. In the continuous search for environmentally friendly crop protection, exploitation of RNA interferance machinery is showing promising results. It is well established that small RNAs (sRNAs) including microRNA (miRNA) and small interfering RNA (siRNA) are involved in the regulation of gene expression via both transcriptional and post-transcriptional RNA silencing. sRNAs from host plants can enter into pathogen cells during invasion and silence pathogen genes. This process has been exploited through Host-Induced Gene Silencing (HIGS), in which plant transgenes that produce sRNAs are engineered to silence pest and pathogen genes. Similarly, exogenously applied sRNAs can enter pest and pathogen cells, either directly or via the hosts, and silence target genes. This process has been exploited in Spray-Induced Gene Silencing (SIGS). Here, we focus on the role of sRNAs and review how they have recently been used against various plant pathogens through HIGS or SIGS-based methods and discuss advantages and drawbacks of these approaches.

Sparking a sulfur war between plants and pathogens.

The biochemical versatility of sulfur (S) lends itself to myriad roles in plant-pathogen interactions. This review evaluates the current understanding of mechanisms by which pathogens acquire S from their plant hosts and highlights new evidence that plants can limit S availability during the immune responses. We discuss the discovery of host disease-susceptibility genes related to S that can be genetically manipulated to create new crop resistance. Finally, we summarize future research challenges and propose a research agenda that leverages systems biology approaches for a holistic understanding of this important element's diverse roles in plant disease resistance and susceptibility.

Recent advances in understanding of fungal and oomycete effectors.

Fungal and oomycete pathogens secrete complex arrays of proteins and small RNAs to interface with plant-host targets and manipulate plant regulatory networks to the microbes' advantage. Research on these important virulence factors has been accelerated by improved genome sequences, refined bioinformatic prediction tools, and exploitation of efficient platforms for understanding effector gene expression and function. Recent studies have validated the expectation that oomycetes and fungi target many of the same sectors in immune signaling networks, but the specific host plant targets and modes of action are diverse. Effector research has also contributed to deeper understanding of the mechanisms of effector-triggered immunity.

In Science Journals.

Highlights from the Science family of journals.

A split green fluorescent protein system to enhance spatial and temporal sensitivity of translating ribosome affinity purification.

Translating ribosome affinity purification (TRAP) utilizes transgenic plants expressing a ribosomal protein fused to a tag for affinity co-purification of ribosomes and the mRNAs that they are translating. This population of actively translated mRNAs (translatome) can be interrogated by quantitative PCR or RNA sequencing. Condition- or cell-specific promoters can be utilized to isolate the translatome of specific cell types, at different growth stages and/or in response to environmental variables. While advantageous for revealing differential expression, this approach may not provide sufficient sensitivity when activity of the condition/cell-specific promoter is weak, when ribosome turnover is low in the cells of interest, or when the targeted cells are ephemeral. In these situations, expressing tagged ribosomes under the control of these specific promoters may not yield sufficient polysomes for downstream analysis. Here, we describe a new TRAP system that employs two transgenes: One is constitutively expressed and encodes a ribosomal protein fused to one fragment of a split green fluorescent protein (GFP); the second is controlled by a stimulus-specific promoter and encodes the second GFP fragment fused to an affinity purification tag. In cells where both transgenes are active, the purification tag is attached to ribosomes by bi-molecular folding and assembly of the split GFP fragments. This approach provides increased sensitivity and better temporal resolution because it labels pre-existing ribosomes and does not depend on rapid ribosome turnover. We describe the optimization and key parameters of this system, and then apply it to a plant-pathogen interaction in which spatial and temporal resolution are difficult to achieve with current technologies.

Long-Term Outcomes of Biotechnology Student Participation in Undergraduate Research Experiences at Delaware Technical Community College.

Engagement in undergraduate research experiences (UREs) positively impacts student skill development, scientific identity, and retention in STEM. Incorporating UREs into 2-year programs would greatly benefit the diverse, nontraditional student populations enrolled at community colleges. This article describes the infusion of the Bioscience/Biotechnology program at Delaware Technical Community College with course-based and mentored research experiences that could serve as a model for other institutions. Studies done with the Office of Institutional Research revealed a concurrent increase in enrollment and graduation rates. Retrospective interviews with graduates from the program highlight the critical influence of research, the mentor/student relationship, and a sense of community on the development of transferable skills, self-efficacy, and subsequent successes in pursuing higher education and employment.

The Arabidopsis PHD-finger protein EDM2 has multiple roles in balancing NLR immune receptor gene expression.

Plant NLR-type receptors serve as sensitive triggers of host immunity. Their expression has to be well-balanced, due to their interference with various cellular processes and dose-dependency of their defense-inducing activity. A genetic "arms race" with fast-evolving pathogenic microbes requires plants to constantly innovate their NLR repertoires. We previously showed that insertion of the COPIA-R7 retrotransposon into RPP7 co-opted the epigenetic transposon silencing signal H3K9me2 to a new function promoting expression of this Arabidopsis thaliana NLR gene. Recruitment of the histone binding protein EDM2 to COPIA-R7-associated H3K9me2 is required for optimal expression of RPP7. By profiling of genome-wide effects of EDM2, we now uncovered additional examples illustrating effects of transposons on NLR gene expression, strongly suggesting that these mobile elements can play critical roles in the rapid evolution of plant NLR genes by providing the "raw material" for gene expression mechanisms. We further found EDM2 to have a global role in NLR expression control. Besides serving as a positive regulator of RPP7 and a small number of other NLR genes, EDM2 acts as a suppressor of a multitude of additional NLR genes. We speculate that the dual functionality of EDM2 in NLR expression control arose from the need to compensate for fitness penalties caused by high expression of some NLR genes by suppression of others. Moreover, we are providing new insights into functional relationships of EDM2 with its interaction partner, the RNA binding protein EDM3/AIPP1, and its target gene IBM1, encoding an H3K9-demethylase.

Iron homeostasis and plant immune responses: Recent insights and translational implications.

Iron metabolism and the plant immune system are both critical for plant vigor in natural ecosystems and for reliable agricultural productivity. Mechanistic studies of plant iron home-ostasis and plant immunity have traditionally been carried out in isolation from each other; however, our growing understanding of both processes has uncovered significant connections. For example, iron plays a critical role in the generation of reactive oxygen intermediates during immunity and has been recently implicated as a critical factor for immune-initiated cell death via ferroptosis. Moreover, plant iron stress triggers immune activation, suggesting that sensing of iron depletion is a mechanism by which plants recognize a pathogen threat. The iron deficiency response engages hormone signaling sectors that are also utilized for plant immune signaling, providing a probable explanation for iron-immunity cross-talk. Finally, interference with iron acquisition by pathogens might be a critical component of the immune response. Efforts to address the global burden of iron deficiency-related anemia have focused on classical breeding and transgenic approaches to develop crops biofortified for iron content. However, our improved mechanistic understanding of plant iron metabolism suggests that such alterations could promote or impede plant immunity, depending on the nature of the alteration and the virulence strategy of the pathogen. Effects of iron biofortification on disease resistance should be evaluated while developing plants for iron biofortification.

Increased Expression of UMAMIT Amino Acid Transporters Results in Activation of Salicylic Acid Dependent Stress Response.

In addition to their role in the biosynthesis of important molecules such as proteins and specialized metabolites, amino acids are known to function as signaling molecules through various pathways to report nitrogen status and trigger appropriate metabolic and cellular responses. Moreover, changes in amino acid levels through altered amino acid transporter activities trigger plant immune responses. Specifically, loss of function of major amino acid transporter, over-expression of cationic amino acid transporter, or over-expression of the positive regulators of membrane amino acid export all lead to dwarfed phenotypes and upregulated salicylic acid (SA)-induced stress marker genes. However, whether increasing amino acid exporter protein levels lead to similar stress phenotypes has not been investigated so far. Recently, a family of transporters, namely USUALLY MULTIPLE ACIDS MOVE IN AND OUT TRANSPORTERS (UMAMITs), were identified as amino acid exporters. The goal of this study was to investigate the effects of increased amino acid export on plant development, growth, and reproduction to further examine the link between amino acid transport and stress responses. The results presented here show strong evidence that an increased expression of UMAMIT transporters induces stress phenotypes and pathogen resistance, likely due to the establishment of a constitutive stress response via a SA-dependent pathway.

Draft Assembly of Phytophthora capsici from Long-Read Sequencing Uncovers Complexity.

Resolving complex plant pathogen genomes is important for identifying the genomic shifts associated with rapid adaptation to selective agents such as hosts and fungicides, yet assembling these genomes remains challenging and expensive. Phytophthora capsici is an important, globally distributed plant pathogen that exhibits widespread fungicide resistance and a broad host range. As with other pathogenic oomycetes, P. capsici has a complex life history and a complex genome. Here, we leverage Oxford Nanopore Technologies and existing short-read resources to rapidly generate a low-cost, improved assembly. We generated 10 Gbp from a single MinION flow cell resulting in >1.25 million reads with an N50 of 13 kb. The resulting assembly is 95.2 Mbp in 424 scaffolds with an N50 length of 313 kb. This assembly is approximately 30 Mbp bigger than the current reference genome of 64 Mbp. We confirmed this larger genome size using flow cytometry, with an estimated size of 110 Mbp. BUSCO analysis identified 97.4% complete orthologs (19.2% duplicated). Evolutionary analysis supports a recent whole-genome duplication in this group. Our work provides a blueprint for rapidly integrating benchtop long-read sequencing with existing short-read data, to dramatically improve assembly quality and integrity of complex genomes and offer novel insights into pathogen genome function and evolution.

Focus on Activation, Regulation, and Evolution of MTI and ETI.

Plants perceive a variety of molecules produced by microbes, insects, and nematodes. These pathogen-derived components include so-called microbe-associated molecular patterns, or MAMPs, as well as effector proteins that are secreted to the exterior or interior of plant cells and these molecules can be recognized by receptor protein complexes on the exterior or interior of plant cells, thereby activating MAMP- or effector-triggered immunity (MTI or ETI, respectively). Because these processes are key components of plant disease resistance, they have been studied intensively. We are now in a golden age of ETI and MTI research, in which mechanistic and evolutionary understanding of both processes is emerging rapidly. Accordingly, in this Focus issue , we explore diverse aspects of MTI and ETI, with a unifying theme of integration at multiple levels.

The Arabidopsis RRM domain protein EDM3 mediates race-specific disease resistance by controlling H3K9me2-dependent alternative polyadenylation of RPP7 immune receptor transcripts.

The NLR-receptor RPP7 mediates race-specific immunity in Arabidopsis. Previous screens for enhanced downy mildew (edm) mutants identified the co-chaperone SGT1b (EDM1) and the PHD-finger protein EDM2 as critical regulators of RPP7. Here, we describe a third edm mutant compromised in RPP7 immunity, edm3. EDM3 encodes a nuclear-localized protein featuring an RNA-recognition motif. Like EDM2, EDM3 promotes histone H3 lysine 9 dimethylation (H3K9me2) at RPP7. Global profiling of H3K9me2 showed EDM3 to affect this silencing mark at a large set of loci. Importantly, both EDM3 and EDM2 co-associate in vivo with H3K9me2-marked chromatin and transcripts at a critical proximal polyadenylation site of RPP7, where they suppress proximal transcript polyadeylation/termination. Our results highlight the complexity of plant NLR gene regulation, and establish a functional and physical link between a histone mark and NLR-transcript processing.

Oomycetes Used in Arabidopsis Research.

Arabidopsis plants in their natural environment are susceptible to infection by oomycete pathogens, in particular to downy mildew and white rust diseases. These naturally occurring infectious agents have imposed evolutionary pressures on Arabidopsis populations and are therefore highly relevant for the study of host-pathogen co-evolution. In addition, the study of oomycete diseases, including infections caused by several Phytophthora species, has led to many scientific discoveries on Arabidopsis immunity and disease. Herein, we describe the major oomycete species used for experiments on Arabidopsis, and how these pathosystems have been used to provide significant insights into mechanistic and evolutionary aspects of plant-oomycete interactions. We also highlight understudied aspects of plant-oomycete interactions, as well as translational approaches, that can be productively addressed using the reference pathosystems described in this article.

Review: Functional linkages between amino acid transporters and plant responses to pathogens.

Upon infection, plant pathogens become dependent on their hosts for nutrition. Therefore, the interaction between the two organisms is tightly linked to the availability and flux of nutrients in the plant. The plant's nitrogen metabolism is reprogrammed during pathogen attack, likely reflecting plant's response to invasion by the pathogen and active modification by the pathogen to promote feeding. Several lines of evidence indicate that plant-derived amino acids are an important source of nitrogen for diverse pathogens. Moreover, amino acid homeostasis is interconnected with the plant's immune signaling pathways. Here, we critically examine the knowns and unknowns about connections between plant-encoded amino acid transporters and resistance or susceptibility to pathogens and pests. We use recent insights into sugar transporters to frame a perspective with potential applicability to amino acids and other nutrients. We emphasize different approaches that have provided insight in this topic and we conclude with suggestions to fill gaps in foundational knowledge and explore new avenues for disease control.

Application of alignment-free bioinformatics methods to identify an oomycete protein with structural and functional similarity to the bacterial AvrE effector protein.

Diverse plant pathogens export effector proteins to reprogram host cells. One of the most challenging goals in the molecular plant-microbe field is to functionally characterize the complex repertoires of effectors secreted by these pathogens. For bacterial pathogens, the predominant class of effectors is delivered to host cells by Type III secretion. For oomycetes, the predominant class of effectors is defined by a signal peptide that mediates secretion from the oomycete and a conserved RxLR motif. Downy mildew pathogens and Phytophthora species maintain hundreds of candidate RxLR effector genes in their genomes. Although no primary sequence similarity is evident between bacterial Type III effectors (T3Es) and oomycete RXLR effectors, some bacterial and oomycete effectors have convergently evolved to target the same host proteins. Such effectors might have evolved domains that are functionally similar but sequence-unrelated. We reasoned that alignment-free bioinformatics approaches could be useful to identify structural similarities between bacterial and oomycete effectors. To test this approach, we used partial least squares regression, alignment-free bioinformatics methods to identify effector proteins from the genome of the oomycete Hyaloperonospora arabidopsidis that are similar to the well-studied AvrE1 effector from Pseudomonas syringae. This approach identified five RxLR proteins with putative structural similarity to AvrE1. We focused on one, HaRxL23, because it is an experimentally validated effector and it is conserved between distantly related oomycetes. Several experiments indicate that HaRxL23 is functionally similar to AvrE1, including the ability to partially rescue an AvrE1 loss-of-function mutant. This study provides an example of how an alignment-free bioinformatics approach can identify functionally similar effector proteins in the absence of primary sequence similarity. This approach could be useful to identify effectors that have convergently evolved regardless of whether the shared host target is known.

Epidermis and Enamel: Insights Into Gnawing Criticisms of Human Bitemark Evidence.

Critics describe forensic dentists' management of bitemark evidence as junk science with poor sensitivity and specificity and state that linkages to a biter are unfounded. Those vocal critics, supported by certain media, characterize odontologists' previous errors as egregious and petition government agencies to render bitemark evidence inadmissible. Odontologists acknowledge that some practitioners have made past mistakes. However, it does not logically follow that the errors of a few identify a systemic failure of bitemark analysis. Scrutiny of the contentious cases shows that most occurred 20 to 40 years ago. Since then, research has been ongoing and more conservative guidelines, standards, and terminology have been adopted so that past errors are no longer reflective of current safeguards. The authors recommend a comprehensive root analysis of problem cases to be used to determine all the factors that contributed to those previous problems. The legal community also shares responsibility for some of the past erroneous convictions. Currently, most proffered bitemark cases referred to odontologists do not reach courts because those forensic dentists dismiss them as unacceptable or insufficient for analysis. Most bitemark evidence cases have been properly managed by odontologists. Bitemark evidence and testimony remain relevant and have made significant contributions in the justice system.

Conserved RxLR Effectors From Oomycetes Hyaloperonospora arabidopsidis and Phytophthora sojae Suppress PAMP- and Effector-Triggered Immunity in Diverse Plants.

Effector proteins are exported to the interior of host cells by diverse plant pathogens. Many oomycete pathogens maintain large families of candidate effector genes, encoding proteins with a secretory leader followed by an RxLR motif. Although most of these genes are very divergent between oomycete species, several genes are conserved between Phytophthora species and Hyaloperonospora arabidopsidis, suggesting that they play important roles in pathogenicity. We describe a pair of conserved effector candidates, HaRxL23 and PsAvh73, from H. arabidopsidis and P. sojae respectively. We show that HaRxL23 is expressed early during infection of Arabidopsis. HaRxL23 triggers an ecotype-specific defense response in Arabidopsis, suggesting that it is recognized by a host surveillance protein. HaRxL23 and PsAvh73 can suppress pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) in Nicotiana benthamiana and effector-triggered immunity (ETI) in soybean. Transgenic Arabidopsis constitutively expressing HaRxL23 or PsAvh73 exhibit suppression of PTI and enhancement of bacterial and oomycete virulence. Together, our experiments demonstrate that these conserved oomycete RxLR effectors suppress PTI and ETI across diverse plant species.