Summary: Frosty pod rot ( FPR ) of T heobroma cacao (cacao) is caused by the hemibiotrophic fungus M oniliophthora roreri . Cacao clones tolerant to FPR are being planted throughout C entral A merica. To determine whether M . roreri shows a differential molecular response during successful infections of tolerant clones, we collected field‐infected pods at all stages of symptomatology for two highly susceptible clones (Pound‐7 and CATIE ‐1000) and three tolerant clones ( UF ‐273, CATIE ‐ R 7 and CATIE ‐ R 4). Metabolite analysis was carried out on clones P ound‐7, CATIE ‐1000, CATIE ‐ R 7 and CATIE ‐ R 4. As FPR progressed, the concentrations of sugars in pods dropped, whereas the levels of trehalose and mannitol increased. Associations between symptoms and fungal loads and some organic and amino acid concentrations varied depending on the clone. RNA ‐Seq analysis identified 873 M . roreri genes that were differentially expressed between clones, with the primary difference being whether the clone was susceptible or tolerant. Genes encoding transcription factors, heat shock proteins, transporters, enzymes modifying membranes or cell walls and metabolic enzymes, such as malate synthase and alternative oxidase, were differentially expressed. The differential expression between clones of 43 M . roreri genes was validated by real‐time quantitative reverse transcription polymerase chain reaction. The expression profiles of some genes were similar in susceptible and tolerant clones (other than CATIE‐R 4) and varied with the biotrophic/necrotropic shift. M oniliophthora roreri genes associated with stress metabolism and responses to heat shock and anoxia were induced early in tolerant clones, their expression profiles resembling that of the necrotrophic phase. M oniliophthora roreri stress response genes, induced during the infection of tolerant clones, may benefit the fungus in overcoming cacao defense mechanisms.

Tropane alkaloids (TAs) are heterocyclic nitrogenous metabolites found across seven orders of angiosperms, including Malpighiales (Erythroxylaceae) and Solanales (Solanaceae). Despite the well-established euphorigenic properties of Erythroxylaceae TAs like cocaine, their biosynthetic pathway remains incomplete. Using yeast as a screening platform, we identified and characterized the missing steps of TA biosynthesis inErythroxylum coca. We first characterize putativeE. cocapolyamine synthase- and amine oxidase-like enzymes in vitro, in yeast, and in planta to show that the first tropane ring closure in Erythroxylaceae occurs via bifunctional spermidine synthase/N-methyltransferases and both flavin- and copper-dependent amine oxidases. We next identify a SABATH family methyltransferase responsible for the 2-carbomethoxy moiety characteristic of Erythroxylaceae TAs and demonstrate that its coexpression with methylecgonone reductase in yeast engineered to express the Solanaceae TA pathway enables the production of a hybrid TA with structural features of both lineages. Finally, we use clustering analysis ofErythroxylumtranscriptome datasets to discover a cytochrome P450 of the CYP81A family responsible for the second tropane ring closure in Erythroxylaceae, and demonstrate the function of the core coca TA pathway in vivo via reconstruction and de novo biosynthesis of methylecgonine in yeast. Collectively, our results provide strong evidence that TA biosynthesis in Erythroxylaceae and Solanaceae is polyphyletic and that independent recruitment of unique biosynthetic mechanisms and enzyme classes occurred at nearly every step in the evolution of this pathway.

The non-climacteric octoploid strawberry (Fragaria × ananassa Duchesne ex Rozier) was used as a model to study its regulation during fruit ripening. High performance liquid chromatography electrospray tandem-mass spectrometry (HPLC-ESI-MS/MS) was employed to profile 28 different endogenous phytohormones in strawberry. These include auxins, cytokinins (CKs), abscisic acid (ABA), ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), jasmonates, and phenolic compounds salicylic acid (SA), benzoic acid (BzA) and phenylacetic acid (PAA) together with their various metabolic forms that have remained largely unexplored thus far. ABA, ACC and CK N⁶-(Δ²-isopentenyl)adenine (iP) were found to be associated with ripening while ABA catabolites 9-hydroxy-ABA and phaseic acid mimicked the pattern of climacteric decline at the turning phase of strawberry ripening. The content of other CK forms except iP decreased as fruit ripened, as also that of auxins indole-3-acetic acid (IAA) and oxo-IAA, and of jasmonates. Data presented here also suggest that both the transition and progression of strawberry fruit ripening are associated with N⁶-(Δ²-isopentenyl)adenosine-5'-monophosphate (iPRMP) → N⁶-(Δ²-isopentenyl)adenosine (iPR) → iP as the preferred CK metabolic pathway. In contrast, the ethylene precursor ACC was present at higher levels, with its abundance increasing from the onset of ripening to the red ripe stage. Further investigation of ripening-specific ACC accumulation revealed the presence of a large ACC synthase (ACS) encoding gene family in octoploid strawberry that was previously unknown. Seventeen ACS genes were found differentially expressed in fruit tissues, while six of them showed induced expression during strawberry fruit ripening. These data suggest a possible role(s) of ACC, ABA, and iP in strawberry fruit ripening. These data add new dimension to the existing knowledge of the interplay of different endogenous phytohormones in octoploid strawberry, paving the way for further investigation of their individual role(s) in fruit ripening.

Main conclusion: Identification of the polyamine biosynthetic pathway genes in duckweed S. polyrhiza reveals presence of prokaryotic as well as land plant-type ADC pathway but absence of ODC encoding genes. Their differential gene expression and transcript abundance is shown modulated by exogenous methyl jasmonate, salinity, and acidic pH. Genetic components encoding for polyamine (PA) biosynthetic pathway are known in several land plant species; however, little is known about them in aquatic plants. We utilized recently sequenced three duckweed (Spirodela polyrhiza) genome assemblies to map PA biosynthetic pathway genes in S. polyrhiza. PA biosynthesis in most higher plants except for Arabidopsis involves two pathways, via arginine decarboxylase (ADC) and ornithine decarboxylase (ODC). ADC-mediated PA biosynthetic pathway genes, namely, one arginase (SpARG1), two arginine decarboxylases (SpADC1, SpADC2), one agmatine iminohydrolase/deiminase (SpAIH), one N-carbamoyl putrescine amidase (SpCPA), three S-adenosylmethionine decarboxylases (SpSAMDc1, 2, 3), one spermidine synthase (SpSPDS1) and one spermine synthase (SpSPMS1) in S. polyrhiza genome were identified here. However, no locus was found for ODC pathway genes in this duckweed. Hidden Markov Model protein domain analysis established that SpADC1 is a prokaryotic/biodegradative type ADC and its molecular phylogenic classification fell in a separate prokaryotic origin ADC clade with SpADC2 as a biosynthetic type of arginine decarboxylase. However, thermospermine synthase (t-SPMS)/Aculis5 genes were not found present. Instead, one of the annotated SPDS may also function as SPMS, since it was found associated with the SPMS phylogenetic clade along with known SPMS genes. Moreover, we demonstrate that S. polyrhiza PA biosynthetic gene transcripts are differentially expressed in response to unfavorable conditions, such as exogenously added salt, methyl jasmonate, or acidic pH environment as well as in extreme temperature regimes. Thus, S. polyrhiza genome encodes for complete polyamine biosynthesis pathway and the genes are transcriptionally active in response to changing environmental conditions suggesting an important role of polyamines in this aquatic plant.

Two apple rootstock genotypes G.935 and B.9 were recently demonstrated to exhibit distinct resistance responses following infection by Pythium ultimum. As part of an effort to elucidate the genetic regulation of apple root resistance to soilborne pathogens, preinoculation transcriptome variations in roots of these two apple rootstock genotypes are hypothesized to contribute to the observed disease resistance phenotypes. Results from current comparative transcriptome analysis demonstrated elevated transcript abundance for many genes which function in a system-wide defense response in the root tissue of the resistant genotype of G.935 in comparison with susceptible B.9. Based on the functional annotation, these differentially expressed genes encode proteins that function in several tiers of defense responses, such as pattern recognition receptors for pathogen detection and subsequent signal transduction, defense hormone biosynthesis and signaling, transcription factors with known roles in defense activation, enzymes of secondary metabolism, and various classes of resistance proteins. The data set suggested a more poised status, which is ready to defend pathogen infection, in the root tissues of resistant genotype of G.935, compared to the susceptible B.9. The significance of preformed defense in the absence of a pathogen toward overall resistance phenotypes in apple root and the potential fitness cost due to the overactivated defense system were discussed.

Phytoplasmas are small, intracellular bacteria that infect a vast range of plant species, causing significant economic losses and impacting agriculture and farmers’ livelihoods. Early and rapid diagnosis of phytoplasma infections is crucial for preventing the spread of these diseases, particularly through early symptom recognition in the field by farmers and growers. A symptom database for phytoplasma infections can assist in recognizing the symptoms and enhance early detection and management. In this study, nearly 35,000 phytoplasma sequence entries were retrieved from the NCBI nucleotide database using the keyword “phytoplasma” and information on phytoplasma disease-associated plant hosts and symptoms was gathered. A total of 945 plant species were identified to be associated with phytoplasma infections. Subsequently, links to symptomatic images of these known susceptible plant species were manually curated, and the Phytoplasma Disease Symptom Database (iPhyDSDB) was established and implemented on a web-based interface using the MySQL Server and PHP programming language. One of the key features of iPhyDSDB is the curated collection of links to symptomatic images representing various phytoplasma-infected plant species, allowing users to easily access the original source of the collected images and detailed disease information. Furthermore, images and descriptive definitions of typical symptoms induced by phytoplasmas were included in iPhyDSDB. The newly developed database and web interface, equipped with advanced search functionality, will help farmers, growers, researchers, and educators to efficiently query the database based on specific categories such as plant host and symptom type. This resource will aid the users in comparing, identifying, and diagnosing phytoplasma-related diseases, enhancing the understanding and management of these infections.