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Rust fungi cause epidemics that threaten the production of important plant species, such as wheat and soy. Melampsora larici-populina (Mlp) causes the poplar rust and encodes at least 1184 candidate effectors (CEs) whose functions are poorly known. In this study, we sequenced the transcriptome and used mass spectrometry to analyze the metabolome of Arabidopsis plants constitutively expressing 14 Mlp CEs and of a control line to discover alterations leading to plant susceptibility. We found 2299 deregulated genes across the experiment. Genes involved in pattern-triggered immunity, such as FRK1, PR1, RBOHD, and WRKY33, as well as AUX/IAA genes were down-regulated. We further observed that 680 metabolites were deregulated in at least one CE-expressing transgenic line, with “highly unsaturated and phenolic compounds” and “peptides” enriched among down- and up-regulated metabolites. Interestingly, transgenic lines expressing unrelated CEs had correlated patterns of gene and metabolite deregulation, while expression of CEs belonging to the same family deregulated different genes and metabolites. Thus, our results uncouple effector sequence similarity and function. This supports that effector functional investigation in the context of their virulence activity and effect on plant susceptibility requires the investigation of the individual effector and precludes generalization based on sequence similarity.

• Salicylic acid (SA) and jasmonic acid (JA) often play distinct roles in plant defence against pathogens. Research from Arabidopsis thaliana has established that SA- and JA-mediated defences are more effective against biotrophs and necrotrophs, respectively. These two hormones often interact antagonistically in response to particular attackers, with the induction of one leading to suppression of the other. Here, we report a contrasting pattern in the woody perennial Populus: positive SA–JA interplay. • Using genetically engineered high SA lines of black poplar and wild-type lines after exogenous hormone application, we quantified SA and JA metabolites, signalling gene transcripts, antifungal flavonoids and resistance to rust (Melampsora larici-populina). • Salicylic acid and JA metabolites were induced concurrently upon rust infection in poplar genotypes with varying resistance levels. Analysis of SA-hyperaccumulating transgenic poplar lines showed increased jasmonate levels, elevated flavonoid content and enhanced rust resistance, but no discernible reduction in growth. Exogenous application of either SA or JA triggered the accumulation of the other hormone. Expression of pathogenesis-related (PR) genes, frequently used as markers for SA signalling, was not correlated with SA content, but rather activated in proportion to pathogen infection. • We conclude that SA and JA pathways interact positively in poplar resulting in the accumulation of flavonoid phytoalexins.

Poplar trees synthesize flavan-3-ols (catechin and proanthocyanidins) as a defense against foliar rust fungi, but the regulation of this defense response is poorly understood. Here, we investigated the role of hormones in regulating flavan-3-ol accumulation in poplar during rust infection. We profiled levels of defense hormones, signaling genes, and flavan-3-ol metabolites in black poplar leaves at different stages of rust infection. Hormone levels were manipulated by external sprays, genetic engineering, and drought to reveal their role in rust fungal defenses. Levels of salicylic acid (SA), jasmonic acid, and abscisic acid increased in rust-infected leaves and activated downstream signaling, with SA levels correlating closely with those of flavan-3- ols. Pretreatment with the SA analog benzothiadiazole increased flavan-3-ol accumulation by activating the MYB–bHLH–WD40 complex and reduced rust proliferation. Furthermore, transgenic poplar lines overproducing SA exhibited higher amounts of flavan-3-ols constitutively via the same transcriptional activation mechanism. These findings suggest a strong association among SA, flavan-3-ol biosynthesis, and rust resistance in poplars. Abscisic acid also promoted poplar defense against rust infection, but likely through stomatal immunity independent of flavan-3-ols. Jasmonic acid did not confer any apparent defense responses to the fungal pathogen. We conclude that SA activates flavan-3-ol biosynthesis in poplar against rust infection.

Differences in Populus nigra L. clone resistance to the rust fungus Melampsora larici-populina Kleb. (MLP) were studied in field trials where infected trees were compared with fungicide-protected trees. MLP rust infections were assessed using a 6-point scale. Four parameters of poplar growth were also measured to gauge host response to infection: shoot thickness, shoot number at the end of the growing season, individual plant dry weight, and dry matter yield per unit area. Five of eight known pathogen virulence types were detected. Cumulative growth in shoot thickness in sprayed and unsprayed plots was similar in clones with high rust pathogen resistance, but significantly different in clones with low resistance. Clones with low resistance also exhibited delayed growth initiation in the year following infection, an effect attributed to lower food storage accumulated during the previous year, reflected in a reduction in stem diameter. Based on stem thickness measurements, it was confirmed growth ceased at the end of August. Average rust severity symptoms ranged from 2.75 to 4.22 on the 6-point scale. The percentage reduction for the various growth parameters resulting from rust infection ranged as follows: individual plant dry weight 5-64%; dry matter yield 21-66%; shoot number 17-46%; and stem diameter 1-35%. Exclusive of stem diameter, these reductions correlated with severity in MLP rust infection. The one exception was tolerance to rust infection displayed by one of the clones. Clones with the lowest growth reductions were 97/152 and 97/157. Growth and yield parameter variation as a result MLP resistance difference was observed among tested P. nigra clones. This variation can be viewed as confirmation that resistance observed in this study and P. nigra clones is a suite of quantitative traits. These findings have important implications for MLP resistance breeding programs.

Plants release complex volatile blends after separate attack by herbivores and pathogens, which play many roles in interactions with other organisms. Large perennials are often attacked by multiple enemies, but the effect of combined attacks on volatile emission is rarely studied, particularly in trees. We infested Populus nigra trees with a pathogen, the rust fungus Melampsora laricipopulina, and Lymantria dispar caterpillars alone and in combination. We investigated poplar volatile emission and its regulation, as well as the behavior of the caterpillars towards volatiles from rust-infected and uninfected trees. Both the rust fungus and the caterpillars alone induced volatile emission from poplar trees. However, the herbivore-induced volatile emission was significantly reduced when trees were under combined attack by the herbivore and the fungus. Herbivory induced terpene synthase transcripts as well as jasmonate concentrations, but these increases were suppressed when the tree was additionally infected with rust. Caterpillars preferred volatiles from rust-infected over uninfected trees. Our results suggest a defense hormone crosstalk upon combined herbivore–pathogen attack in poplar trees which results in lowered emission of herbivore-induced volatiles. This influences the preference of herbivores, and might have other far-reaching consequences for the insect and pathogen communities in natural poplar forests.

Rust fungi are some of the most devastating pathogens of crop plants. They are obligate biotrophs, which extract nutrients only from living plant tissues and cannot grow apart from their hosts. Their lifestyle has slowed the dissection of molecular mechanisms underlying host invasion and avoidance or suppression of plant innate immunity. We sequenced the 101-Mb genome of Melampsora larici-populina, the causal agent of poplar leaf rust, and the 89-Mb genome of Puccinia graminis f. sp. tritici, the causal agent of wheat and barley stem rust. We then compared the 16,399 predicted proteins of M. larici-populina with the 17,773 predicted proteins of P. graminis f. sp tritici. Genomic features related to their obligate biotrophic lifestyle include expanded lineage-specific gene families, a large repertoire of effector-like small secreted proteins, impaired nitrogen and sulfur assimilation pathways, and expanded families of amino acid and oligopeptide membrane transporters. The dramatic up-regulation of transcripts coding for small secreted proteins, secreted hydrolytic enzymes, and transporters in planta suggests that they play a role in host infection and nutrient acquisition. Some of these genomic hallmarks are mirrored in the genomes of other microbial eukaryotes that have independently evolved to infect plants, indicating convergent adaptation to a biotrophic existence inside plant cells.

The aims were to explore the use of wild populations of Populus nigra for breeding and to compare crosses made between genotypes from different areas with different climates. Genetic distances ranged from 0.175 (populations growing under optimal conditions) up to 0.875 (populations growing under marginal conditions). A total of 39 trees were chosen from these populations, and in four different years 58 different parental crosses were made, from which a total of 2,277 individuals were produced for assessment as potential biomass producers. The progenies from each cross were rated according to a selection index (SI) based on three characteristics (height, leading shoot diameter and resistance to the rust Melampsora larici-populina). Sixteen of such crosses had significantly higher SI compared to the lowest SI values observed. In 19 of the crosses, it was shown that certain individual trees had made a significant contribution to lower susceptibility to rust, and in particular, two male trees used in the parental crosses were implicated as being the source of rust resistance. Selected individuals were cloned and used in a field trial with NE-42, as a control. It was found that six clones had significantly higher SI than NE-42. In crosses between trees originating from contrasting conditions, there was a higher average SI than in crosses between trees from the same conditions. However, these differences were not statistically significant. The successful clones are recommended for use in those areas where the native populations of pure black poplar are threatened by cross-pollination with allochthonous species.

Melampsora larici-populina causes serious poplar foliar diseases called rust worldwide. Salicylic acid (SA) and jasmonic acid (JA) are important phytohormones that are related to plant defence responses. To investigate the transcriptome profiles of SA- and JA-related genes involved in poplar rust interaction, two tolerant poplars and one intolerant poplar were selected for this study. Weighted gene coexpression network analysis (WGCNA) was applied to characterize the changes in the transcriptome profiles and contents of SA and JA after infection with the virulent E4 race of M. larici-populina. In response to infection with the E4 race of M. larici-populina, tolerant symptoms were correlated with the expression of genes related to SA and JA biosynthesis, the levels of SA and JA, and the expression of defence-related genes downstream of SA and JA. Tolerant poplars could promptly regulate the occurrence of defence responses by activating or inhibiting SA or JA pathways in a timely manner, including regulating the expression of genes related to programmed cell death, such as Kunitz-type trypsin inhibitor (KTI), to limit the growth of E4 and protect themselves. WGCNA suggested that KTI might be regulated by a Cytochrome P450 family (CYP) gene. Some CYPs should play an important role in both JA- and SA-related pathways. In contrast, in intolerant poplar, the inhibition of SA-related defence signalling through increasing JA levels in the early stage led to continued inhibition of a large number of plant–pathogen interaction-related and signalling-related genes, including NBS-LRRs, EDS1, NDR1, WRKYs, and PRs. Therefore, timely activation or inhibition of the SA or JA pathways is the key difference between tolerant and intolerant poplars.

Summary Parasite effector proteins target various host cell compartments to alter host processes and promote infection. How effectors cross membrane‐rich interfaces to reach these compartments is a major question in effector biology. Growing evidence suggests that effectors use molecular mimicry to subvert host cell machinery for protein sorting. We recently identified chloroplast‐targeted protein 1 (CTP1), a candidate effector from the poplar leaf rust fungus Melampsora larici‐populina that carries a predicted transit peptide and accumulates in chloroplasts and mitochondria. Here, we show that the CTP1 transit peptide is necessary and sufficient for accumulation in the stroma of chloroplasts. CTP1 is part of a Melampsora‐specific family of polymorphic secreted proteins. Two members of that family, CTP2 and CTP3, also translocate in chloroplasts in an N‐terminal signal‐dependent manner. CTP1, CTP2 and CTP3 are cleaved when they accumulate in chloroplasts, while they remain intact when they do not translocate into chloroplasts. Our findings reveal that fungi have evolved effector proteins that mimic plant‐specific sorting signals to traffic within plant cells. Parasite effector proteins target various host cell compartments to alter host processes and promote infection. Petre et al. show that the poplar leaf rust fungus fungi secretes an effector protein that mimics a plant‐specific sorting signal to traffic into the chloroplasts of host cells.

Populus species are crucial for forestry and industry, yet poplar rust limits their growth. The molecular basis of rust resistance remains poorly understood. This study identified 203 AP2/ERF genes in the Populus trichocarpa genome, clustring into four subfamilies: ERF (169 members), AP2 (29), RAV (5), and Soloist (1), adding 3 ERF subfamily members to previous studies. Promoter cis-acting element analysis revealed that AP2/ERF genes potentially involved in poplar stress responses include 91 for drought, 76 for defense, 62 for low temperature, and 8 for wounding. Additionally, 172, 133, and 98 genes may respond to SA, MeJA, and ABA, respectively. Segmental duplications were the main driver of the expansion of these genes. Promoter regions contained elements linked to stress and hormone responses. Following Melampsora larici-populina ( Mlp ) infection, 74 AP2/ERF genes showed differential expression, forming three modules: purple, yellow, and red. The purple module was more expressed in susceptible Populus purdomii , while the red module was upregulated in resistant poplar, P. szechuanica . The yellow module showed no notable interspecific expression differences. Four ERF genes ( ERF49 , ERF62 , ERF109 , and ERF117 ) from the red module, with significant expression variation, were cloned for sequencing and tissue-specific analysis. Overexpression of these genes in Nicotiana benthamiana increased the transcript levels of defense genes NbPR1 , NbPAL , and NbSOD , boosting tolerance to Sclerotinia sclerotiorum . These results offer key insights into the roles and regulatory mechanisms of AP2/ERF transcription factors in popular species.