Validation of an Alternative Small Stem Assay for Blight Resistance in Chestnut Seedlings and Recommendations for Broader Use.

We evaluated an alternative small stem assay (AltSSA) for blight resistance in backcross hybrid chestnut trees (Castanea dentata/mollissima). Whereas standard small stem assays (SSAs) are done by inoculating small incisions in stems, in our AltSSA, 4- to 5-mm stems are cut off, and the exposed (living) stem tips are inoculated with discs of Cryphonectria parasitica inoculum and temporarily covered with plastic sleeves. Intended primarily for forward selection, this method was designed to be easy to implement, to consistently induce cankering, and to better enable seedling recovery via the development of lateral shoots from the lower stem. After 90+ days, cankers are evaluated and removed, and seedlings are prepared for out-planting. Previous results showed that AltSSAs performed at least as well as a common SSA method in distinguishing resistant and susceptible types. In this follow-up analysis of 35 lines of backcross seedlings studied in 2020 and 2021, we showed that mean orange zone canker length (OZCL) and a multifactor principal components analysis-based blight resistance index gave results consistent with predictions derived from two methods of blight resistance phenotyping and percentage of American chestnut ancestry of the parents of each line. As expected, based upon the apparent polygenic inheritance of blight resistance in backcross chestnut trees, mean OZCL of backcross families ranged from intermediate (F1 hybrid-level) to low (wild-type American chestnut-level). Consistent with prior results, canker production was near 100%, survivorship after out-planting was very high, and postinoculation stem dieback was not apparently related to the stem tip inoculations. Altogether, these results suggest that the AltSSA is a viable method for early detection of relative blight resistance in seedlings and may enable a reduction in the numbers of trees out-planted and placed under care for long-term evaluation and breeding. Thus, the AltSSA can prevent time, resources, and orchard space from being used on susceptible trees.

A KARRIKIN INSENSITIVE2 paralog in lettuce mediates highly sensitive germination responses to karrikinolide.

Karrikins (KARs) are chemicals in smoke that can enhance germination of many plants. Lettuce (Lactuca sativa) cv. Grand Rapids germinates in response to nanomolar karrikinolide (KAR1). Lettuce is much less responsive to KAR2 or a mixture of synthetic strigolactone analogs, rac-GR24. We investigated the molecular basis of selective and sensitive KAR1 perception in lettuce. The lettuce genome contains two copies of KARRIKIN INSENSITIVE2 (KAI2), which in Arabidopsis (Arabidopsis thaliana) encodes a receptor that is required for KAR responses. LsKAI2b is more highly expressed than LsKAI2a in dry achenes and during early stages of imbibition. Through cross-species complementation assays in Arabidopsis, we found that an LsKAI2b transgene confers robust responses to KAR1, but LsKAI2a does not. Therefore, LsKAI2b likely mediates KAR1 responses in lettuce. We compared homology models of KAI2 proteins from lettuce and a fire-follower, whispering bells (Emmenanthe penduliflora). This identified pocket residues 96, 124, 139, and 161 as candidates that influence the ligand specificity of KAI2. Further support for the importance of these residues was found through a broader comparison of pocket residues among 281 KAI2 proteins from 184 asterid species. Almost all KAI2 proteins had either Tyr or Phe identity at position 124. Genes encoding Y124-type KAI2 are more broadly distributed in asterids than in F124-type KAI2. Substitutions at residues 96, 124, 139, and 161 in Arabidopsis KAI2 produced a broad array of responses to KAR1, KAR2, and rac-GR24. This suggests that the diverse ligand preferences observed among KAI2 proteins in plants could have evolved through relatively few mutations.

Genome Sequence of Striga asiatica Provides Insight into the Evolution of Plant Parasitism.

Parasitic plants in the genus Striga, commonly known as witchweeds, cause major crop losses in sub-Saharan Africa and pose a threat to agriculture worldwide. An understanding of Striga parasite biology, which could lead to agricultural solutions, has been hampered by the lack of genome information. Here, we report the draft genome sequence of Striga asiatica with 34,577 predicted protein-coding genes, which reflects gene family contractions and expansions that are consistent with a three-phase model of parasitic plant genome evolution. Striga seeds germinate in response to host-derived strigolactones (SLs) and then develop a specialized penetration structure, the haustorium, to invade the host root. A family of SL receptors has undergone a striking expansion, suggesting a molecular basis for the evolution of broad host range among Striga spp. We found that genes involved in lateral root development in non-parasitic model species are coordinately induced during haustorium development in Striga, suggesting a pathway that was partly co-opted during the evolution of the haustorium. In addition, we found evidence for horizontal transfer of host genes as well as retrotransposons, indicating gene flow to S. asiatica from hosts. Our results provide valuable insights into the evolution of parasitism and a key resource for the future development of Striga control strategies.

Structural modelling and transcriptional responses highlight a clade of PpKAI2-LIKE genes as candidate receptors for strigolactones in Physcomitrella patens.

MAIN CONCLUSION: A set of PpKAI2 - LIKE paralogs that may encode strigolactone receptors in Physcomitrella patens were identified through evolutionary, structural, and transcriptional analyses, suggesting that strigolactone perception may have evolved independently in basal land plants in a similar manner as spermatophytes. Carotenoid-derived compounds known as strigolactones are a new class of plant hormones that modulate development and interactions with parasitic plants and arbuscular mycorrhizal fungi. The strigolactone receptor protein DWARF14 (D14) belongs to the α/ß hydrolase family. D14 is closely related to KARRIKIN INSENSITIVE2 (KAI2), a receptor of smoke-derived germination stimulants called karrikins. Strigolactone and karrikin structures share a butenolide ring that is necessary for bioactivity. Charophyte algae and basal land plants produce strigolactones that influence their development. However phylogenetic studies suggest that D14 is absent from algae, moss, and liverwort genomes, raising the question of how these basal plants perceive strigolactones. Strigolactone perception during seed germination putatively evolved in parasitic plants through gene duplication and neofunctionalization of KAI2 paralogs. The moss Physcomitrella patens shows an increase in KAI2 gene copy number, similar to parasitic plants. In this study we investigated whether P. patens KAI2-LIKE (PpKAI2L) genes may contribute to strigolactone perception. Based on phylogenetic analyses and homology modelling, we predict that a clade of PpKAI2L proteins have enlarged ligand-binding cavities, similar to D14. We observed that some PpKAI2L genes have transcriptional responses to the synthetic strigolactone GR24 racemate or its enantiomers. These responses were influenced by light and dark conditions. Moreover, (+)-GR24 seems to be the active enantiomer that induces the transcriptional responses of PpKAI2L genes. We hypothesize that members of specific PpKAI2L clades are candidate strigolactone receptors in moss.

PLANT EVOLUTION. Convergent evolution of strigolactone perception enabled host detection in parasitic plants.

Obligate parasitic plants in the Orobanchaceae germinate after sensing plant hormones, strigolactones, exuded from host roots. In Arabidopsis thaliana, the α/ß-hydrolase D14 acts as a strigolactone receptor that controls shoot branching, whereas its ancestral paralog, KAI2, mediates karrikin-specific germination responses. We observed that KAI2, but not D14, is present at higher copy numbers in parasitic species than in nonparasitic relatives. KAI2 paralogs in parasites are distributed into three phylogenetic clades. The fastest-evolving clade, KAI2d, contains the majority of KAI2 paralogs. Homology models predict that the ligand-binding pockets of KAI2d resemble D14. KAI2d transgenes confer strigolactone-specific germination responses to Arabidopsis thaliana. Thus, the KAI2 paralogs D14 and KAI2d underwent convergent evolution of strigolactone recognition, respectively enabling developmental responses to strigolactones in angiosperms and host detection in parasites.

Evidence that KARRIKIN-INSENSITIVE2 (KAI2) Receptors may Perceive an Unknown Signal that is not Karrikin or Strigolactone.

The α/ß-hydrolases KAI2 and D14 are paralogous receptors for karrikins and strigolactones, two classes of plant growth regulators with butenolide moieties. KAI2 and D14 act in parallel signaling pathways that share a requirement for the F-box protein MAX2, but produce distinct growth responses by regulating different members of the SMAX1-LIKE/D53 family. kai2 and max2 mutants share seed germination, seedling growth, leaf shape, and petiole orientation phenotypes that are not found in d14 or SL-deficient mutants. This implies that KAI2 recognizes an unknown, endogenous signal, herein termed KAI2 ligand (KL). Recent studies of ligand-specificity among KAI2 paralogs in basal land plants and root parasitic plants suggest that karrikin and strigolactone perception may be evolutionary adaptations of KL receptors. Here we demonstrate that evolutionarily conserved KAI2c genes from two parasite species rescue multiple phenotypes of the Arabidopsis kai2 mutant, unlike karrikin-, and strigolactone-specific KAI2 paralogs. We hypothesize that KAI2c proteins recognize KL, which could be an undiscovered hormone.